Method of prophylaxis or treatment of antigen presenting cell driven skin conditions using inhibitors of the CD2/LFA-3 interaction

ABSTRACT

Methods of using inhibitors of the CD2/LFA-3 interaction in treating skin conditions characterized by increased T cell activation and abnormal antigen presentation in the dermis and epidermis in mammals, including humans. Such conditions include psoriasis, UV damage, e.g., photoaging, atopic dermatitis, cutaneous T cell lymphoma such as mycosis fungoides, allergic and irritant contact dermatitis, lichen planus, alopecia areata, pyoderma gangrenosum, vitiligo, ocular cicatricial pemphigoid, and urticaria.

[0001] This application is a continuation-in-part of U.S. Ser. No.07/862,022, filed Apr. 2, 1992 and of PCT/U.S. 92/08755, filed Oct. 6,1992, which is a continuation-in-part of U.S. Ser. No. 07/770,969, filedOct. 7, 1991, all of which are herein incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

[0002] This invention relates to methods of using inhibitors of theCD2/LFA-3 interaction in treating skin conditions characterized byincreased T cell activation and abnormal antigen presentation in thedermis and epidermis in mammals, including humans. Such conditionsinclude psoriasis, UV damage, e.g., photoaging, atopic dermatitis,cutaneous T cell lymphoma such as mycosis fungoides, allergic andirritant contact dermatitis, lichen planus, alopecia areata, pyodermagangrenosum, vitiligo, ocular cicatricial pemphigoid, and urticaria.

BACKGROUND OF THE INVENTION

[0003] There are numerous skin conditions characterized by increased Tcell activation and abnormal antigen presentation in the dermis andepidermis. The pathophysiologic mechanisms involved in the evolution ofsuch inflammatory processes are poorly understood. However, it hasbecome apparent that skin cells are important in the generation of acutaneous inflammatory response (Kupper, “Immune and InflammatoryProcesses in Cutaneous Tissues”, J. Clin. Invest., 86, pp. 1783-89(1990)).

[0004] The normal adult epidermal population contains 1-2% Langerhans'cells and about 98% keratinocytes. Keratinocytes and othernonhematopoietically-derived cells resident in skin contribute to immunehomeostasis and can produce various cytokines which influence migrationof T cells and expression of adhesion molecules.

[0005] As antigen presenting cells, Langerhans' cells express a highdensity of Class II major histocompatibility complex (MHC) antigen onthe cell surface. MHC Class II molecules bind peptides derived fromendocytosed antigen and are recognized primarily by helper Tlymphocytes. The T cell receptor on T cells recognizes antigen as apeptide fragment bound to the cell-surface molecules encoded by the MHC(Springer, “Adhesion Receptors of the Immune System”, Nature, 346, pp.425-27 (1990)).

[0006] There are many interactions between molecules expressed on thesurface of Langerhans' cells and the surface of T cells, in addition tothe T cell receptor/MHC interaction. These surface molecules, oftenreferred to as adhesion molecules, participate in a number of functionsincluding cellular adhesion, antigen recognition, co-stimulatorysignalling in T cell activation and stimulation of effectors of T cellcytotoxicity (“Adhesion Molecules in Diagnosis and Treatment ofInflammatory Diseases”, The Lancet 336, pp. 1351-52 (1990)). Such celladhesion appears to be involved in activation of T cell proliferation inthe generation of an immune response (Hughes et al., “The EndothelialCell as a Regulator of T-cell Function”, Immunol. Rev., 117, pp. 85-102(1990)).

[0007] Various skin conditions are characterized by increased T cellactivation and abnormal antigen presentation in the dermis and epidermis(Cooper, “Immunoregulation in the Skin”, in Cutaneous Lymphoma, Curr.Probl. Dermatol., eds. van Vloten et al., 19, pp. 69-80 at pp. 73, 74,76 (1990)). For example, in contact allergic dermatitis, activation ofintracutaneous T cells is observed. It is known that skin from patientsexhibiting atopic dermatitis contains an increased number of Langerhans'cells (Cooper, “Immunoregulation in the Skin”, in Cutaneous Lymphoma,Curr. Probl. Dermatol., eds. van Vloten et al., 19, at p. 74 (1990)). Inpsoriatic skin, there is an increased number of antigen presentingcells, composed of both Langerhans' cells and non-Langerhans' cell ClassII MHC-bearing antigen presenting cells (Cooper, “Immunoregulation inthe Skin”, in Cutaneous Lymphoma, Curr. Probl. Dermatol., eds. vanVloten et al., 19, at p. 75 (1990)).

[0008] UV exposed skin is characterized by an overall depletion ofLangerhans' cells and migration of a non-Langerhans' cellantigen-presenting cell population into the epidermis, which activatesautologous T cells to proliferate (Cooper, “Immunoregulation in theSkin” in Cutaneous Lymphoma, Curr. Probl. Dermatol., eds. van Vloten etal., 19, at pp. 75-76 (1990)). In human skin after 4 minimal erythemaldoses of UV B, Langerhans' cells (the constitutive antigen presentingcell population) are inactivated for approximately 3 days (Cooper etal., “Effects Of Ultraviolet Radiation On Human Epidermal CellAlloantigen Presentation: Initial Depression Of LangerhansCell-Dependent Function Is Followed By Appearance Of T6-DR⁺ Cells ThatEnhance Epidermal Alloantigen Presentation”, J. Immunol., 134, pp.129-37 (1985)). In this type of UV damaged skin, the CD1a⁻DR⁺ macrophagepopulation (a population of antigen presenting cells) increases from 0%(normal skin) to approximately 2-10% of the entire epidermal cellpopulation and is the cell population entirely responsible for theinduction of T cell proliferation to alloantigen. (Cooper et al., J.Immunol., supra (1985); Baadsgaard et al., “In Vivo Ultraviolet-ExposedHuman Epidermal Cells Activate T Suppressor Cell Pathways That InvolveCD4⁺ CD45RA⁺ Suppressor-Inducer T Cells”, J. Immunol., 145, pp. 2854-61(1990)).

[0009] Cutaneous T cell lymphoma is characterized by the expansion of amalignant clonal population of T cells in the dermis and epidermis.Lesional epidermal cells contain increased numbers of CD1⁺DR⁺ antigenpresenting cells (Cooper, “Immunoregulation in the Skin” in CutaneousLymphoma, Curr. Probl. Dermatol., eds. van Vloten et al., 19, at pp.76-77 (1990)).

[0010] Presently known therapies for the above mentioned skin diseasesare inadequate. Steroids or cyclosporin A are commonly used in thetreatment of psoriasis, lichen planus, urticaria, atopic dermatitis, UVdamage, pyoderma gangrenosum, vitiligo, ocular cicatricial pemphigoid,alopecia areata, allergic and irritant contact dermatitis and cutaneousT cell lymphoma. In addition, for some of these skin conditions, varioustherapies include retinoids, PUVA, nitrogen mustard, interferon,chemotherapy, methotrexate, UV light, antibiotics and antihistamines.See generally Fitzpatrick, Dermatology in General Medicine, 3rd ed.,McGraw Hill (1987).

[0011] Side effects to these therapies are known. Most commonlyencountered drawbacks for cyclosporin A include toxicity due toimmunosuppression and renal and neural toxicity. Steroids have wellknown side effects including induction of Cushing Syndrome. Side effectsof certain of the other aforementioned therapies include skin cancer,bone marrow and constitutional toxicities, ligament calcification, liverfibrosis and other disorders.

[0012] T cells play a major role in the immune response by interactingwith target and antigen presenting cells. For example, T cell-mediatedkilling of target cells is a multi-step process involving, initially,adhesion of cytolytic T cells (the effector cells) to target cells.Also, helper T cells help initiate the immune response by adhesion toantigen presenting cells.

[0013] These interactions of T cells with target and antigen presentingcells are highly specific and depend on the recognition of an antigen onthe surface of a target or antigen presenting cell by one of the manyspecific antigen receptors on the surface of T cells.

[0014] The receptor-antigen interaction of T cells and other cells isalso facilitated by various T cell surface proteins, e.g., theantigen-receptor complex CD3 and accessory adhesion molecules such asCD4, LFA-1, CD8, and CD2. It is also facilitated by accessory adhesionmolecules, such as LFA-3, ICAM-1 and MHC, that are expressed on thesurface of the target or antigen presenting cells. For example, LFA-1and its counter receptor ICAM-1 or ICAM-2,as well as CD2 and its counterreceptor LFA-3 have been implicated in cellular adhesion and T cellactivation. It is known that the LFA-1/ICAM and CD2/LFA-3 interactionsare independent.

[0015] A number of other molecules present on resting T cells have alsobeen implicated in T cell adhesion, including E2 (MIC2), VLA4 (CD49d),CD44 (Hermes, Pgp-1, ECMRIII), and H19 (N4) (see Makgoba et al., “TheCD2-LFA-3 and LFA-1-ICAM Pathways: Relevance to T-cell Recognition”,Immunol. Today, 10, pp. 417-22 (1989)).

[0016] One way in which T cells are activated is by binding of theirantigen specific T cell receptors to peptide-MHC complexes on thesurface of antigen presenting cells such as macrophages. T cellactivation stimulates proliferation and differentiation of two types offunctional T cells: helper cells, which promote the proliferation andmaturation of antibody-producing B lymphocytes, and killer cells, whichlyse target cells (Bierer et al., “A Monoclonal Antibody to LFA-3, theCD2 Ligand, Specifically Immobilizes Major Histocompatibility ComplexProteins”, Eur. J. Immunol., 19, pp. 661-65 (1989); Springer “AdhesionReceptors of the Immune System”, Nature, 346, pp.425-34 (1990)).

[0017] The interaction between CD2 and LFA-3 remains poorly understoodwith respect to activation of T cell activity. Recent studies havesuggested that there is a specific interaction between CD2 (a T celladhesion molecule) and LFA-3 (a target cell and antigen presenting celladhesion molecule) which mediates T cell adhesion to the target orantigen presenting cells. This cell-cell adhesion has been implicated inthe initiation of T cell functional responses (Dustin et al., “PurifiedLymphocyte Function Associated Antigen 3 Binds to CD2 and Mediates TLymphocyte Adhesion,” J. Exp. Med., 165, pp. 677-92 (1987); Springer etal., “The Lymphocyte Function-associated LFA-1, CD2, and LFA-3Molecules: Cell Adhesion Receptors of the Immune System”, Ann. Rev.Immunol., 5, pp. 223-52 (1987)).

[0018] LFA-3, which is found on the surface of a wide variety of cells,including human erythrocytes, has become the subject of a considerableamount of study to further elucidate its role in various T cellinteractions (see, e.g., Krensky et al., “The Functional Significance,Distribution, and Structure of LFA-1, LFA-2, and LFA-3: Cell SurfaceAntigen Associated with CTL-Target Interactions”, J. Immunol., 131(2),pp. 611-16 (1983); Shaw et al., “Two Antigen-Independent AdhesionPathways Used by Human Cytotoxic T-cell Clones”, Nature, 323, pp. 262-64(1986)). Two natural forms of LFA-3 have been identified. One form ofLFA-3 (“transmembrane LFA-3”) is anchored in the cell membrane by atransmembrane hydrophobic domain. cDNA encoding this form of LFA-3 hasbeen cloned and sequenced (see, e.g., Wallner et al., “Primary Structureof Lymphocyte Function-Associated Antigen-3 (LFA-3)”, J. Exp. Med., 166,pp. 923-32 (1987)). Another form of LFA-3 is anchored to the cellmembrane via a covalent linkage to phosphatidylinositol(“PI”)-containing glycolipid. This latter form has been designated“PI-linked LFA-3”, and cDNA encoding this form of LFA-3 has also beencloned and sequenced (Wallner et al., PCT Publn. WO 90/02181).

[0019] The human CD2 (T11) molecule is a 50 kD surface glycoproteinexpressed on >95% of thymocytes and virtually all peripheral Tlymphocytes. Biochemical analyses using specific monoclonal antibodieshave suggested that CD2 is T lineage-specific and exists on the cellsurface in several differentially glycosylated forms (Howard et al., “AHuman T Lymphocyte Differentiation Marker Defined by MonoclonalAntibodies that Block E-Rosette Formation”, J. Immunol., 126, pp.2117-22 (1981); Brown et al., in Leuko Typing III, ed. McMichael, OxfordUniversity Press, pp. 110-12 (1987); Sayre et al., “Molecular Cloningand Expression of T11 cDNAs Reveals a Receptor-Like Structure on Human TLymphocytes”, Proc. Natl. Acad. Sci. USA, 84, pp. 2941-45 (1987)).

[0020] The sequence of a human CD2 gene has been reported (Seed andAruffo, “Molecular Cloning of the CD2 Antigen, the T-cell ErythrocyteReceptor, by a Rapid Immunoselection Procedure”, Proc. Natl. Acad. Sci.USA, 84, pp. 3365-69 (1987); Sayre et al., “Molecular Cloning andExpression of T11 cDNAs Reveal a Receptor-like Structure on Human TLymphocytes”, Proc. Natl. Acad. Sci. USA, 84, pp. 2941-45 (1987)). CD2cDNA clones predict a cleaved signal peptide of 24 amino acid residues,an extracellular segment of 185 residues, a transmembrane domain of 25residues and a cytoplasmic region of 117 residues (Sayre et al., supra(1987); Sewell et al., “Molecular Cloning of the Human T-LymphocyteSurface CD2 (T11) Antigen”, Proc. Natl. Acad. Sci. USA, 83, pp. 8718-22(1986); Seed and Aruffo, supra (1987); Clayton et al., Eur. J. Immunol.,17, pp. 1367-70 (1987)).

[0021] Soluble CD2 polypeptides having an LFA-3 binding domain have beenreported (PCT Publn. WO 90/08187).

[0022] Monoclonal antibodies to CD2, for example TS2/18, T11₁, T11₂,T11₃, and to LFA-3, for example TS2/9, have also been reported (see,e.g., Hughes et al., “The Endothelial Cell as a Regulator of T-CellFunction”, Immunol. Reviews, 117, pp. 85-102 (1990); Meuer, “AnAlternative Pathway of T-Cell Activation: A Functional Role for the 50kD T11 Sheep Erythrocyte Receptor Protein”, Cell, 36, pp. 897-906(1984)).

[0023] The need still exists for improved methods of preventing andtreating skin conditions exhibiting increased T cell activation andabnormal antigen presentation.

SUMMARY OF THE INVENTION

[0024] The present invention generally solves many of the problemsreferred to above. It for the first time provides a method of preventingor treating skin conditions, characterized by increased T cellactivation and abnormal antigen presentation in the dermis andepidermis, in a mammal, whereby an inhibitor of the CD2/LFA-3interaction, is administered to the mammal. The methods of thisinvention are superior to previously available therapies for these skinconditions for many reasons, including less immunosuppression thanpre-existing therapies and more specific therapy with less generaltoxicity.

[0025] The method of the present invention preferably will be used inthe treatment or prophylaxis of skin conditions selected from psoriasis,UV damage, e.g., photoaging, atopic dermatitis, cutaneous T celllymphoma such as mycosis fungoides, allergic and irritant contactdermatitis, lichen planus, alopecia areata, pyoderma gangrenosum,vitiligo, ocular cicatricial pemphigoid, and urticaria, preferablypsoriasis or UV damage.

[0026] Inhibitors that can be used in accordance with the method of thepresent invention include any molecule that inhibits the CD2/LFA-3interaction. Preferably, the inhibitor is selected from the groupconsisting of anti-LFA-3 antibody homologs, anti-CD2 antibody homologs,soluble LFA-3 polypeptides, small molecules, e.g., carbohydrates,soluble CD2 polypeptides, CD2 or LFA-3 mimetic agents and derivativesthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 illustrates the percent inhibition caused by an anti-LFA-3monoclonal antibody (7A6) or an anti-CD2 monoclonal antibody (TS2/18) ascompared to a non-specific control IgG₁ antibody (MOPC21) of autologousT cell activation by psoriatic epidermal cells in 4 patients.

[0028]FIG. 2 illustrates the inhibition of allogeneic T cell activationby UV damaged epidermal cells ([³H]TdR incorporation) caused by ananti-LFA-3 monoclonal antibody (1E6) or an anti-CD2 monoclonal antibody(TS2/18) as compared to a non-specific IgG antibody (MOPC21).

[0029]FIG. 3 illustrates the inhibition of autologous mononuclear cellresponses to psoriatic epidermal cells ([³H]TdR incorporation) caused byan LFA3TIP fusion molecule as compared to a human IgG control.

[0030]FIG. 4 illustrates the effect of LFA3TIP on spontaneous lesionalpsoriatic dermal cell proliferation. A reduction in spontaneous dermalcell proliferation ([³H]TdR incorporation) was seen with dose responsesbetween 0.03 and 0.003 μg/ml of LFA3TIP. LFA3TTP cultures areconsistently less proliferative than the human IgG control culturesthrough the concentration of 0.01 μg/ml.

[0031]FIG. 5 illustrates the inhibition of allogeneic mononuclear cellresponses to normal epidermal cells ([³H]TdR incorporation) caused by anLFA3TIP fusion molecule as compared to a human IgG control.

[0032]FIG. 6 illustrates the inhibition of autologous mononuclear cellresponses to psoriatic dermal cells ([³H]TdR incorporation) caused by anLFA3TIP fusion molecule as compared to a human IgG control.

[0033]FIG. 7 illustrates the inhibition of inflammatory macrophage APCactivity in UV-exposed epidermal cells (EC) ([³H]TdR incorporation)caused by an LFA3TIP fusion molecule as compared to a human IgG control.Two concentrations of UV-EC are shown in panel 7A and 7B. LFA3TIPincubation resulted in approximately 50% inhibition.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Definitions

[0035] As used herein, “CD2” means a CD2 polypeptide that binds to anaturally occurring LFA-3 polypeptide and which is encoded by (a) anaturally occurring mammalian CD2 DNA sequence (e.g., SEQ ID NO:5); (b)a DNA sequence degenerate to a naturally occurring CD2 DNA sequence; or(c) a DNA sequence that hybridizes to one of the foregoing DNA sequencesunder conditions equivalent to about 20° C. to 27° C. below T_(m) and 1M sodium chloride.

[0036] As used herein, “LFA-3” means an LFA-3 polypeptide that binds toa naturally occurring CD2 polypeptide and which is encoded by (a) anaturally occurring mammalian LFA-3 DNA sequence (e.g., SEQ ID NO:1 orSEQ ID NO:3); (b) a DNA sequence degenerate to a naturally occurringLFA-3 DNA sequence; or (c) a DNA sequence that hybridizes to one of theforegoing DNA sequences under conditions to about 20° C. to 27° C. belowT_(m) and 1 M sodium chloride.

[0037] As used herein, a “soluble LFA-3 polypeptide” or a “soluble CD2polypeptide” is an LFA-3 or CD2 polypeptide incapable of anchoringitself in a membrane. Such soluble polypeptides include, for example,CD2 and LFA-3 polypeptides that lack a sufficient portion of theirmembrane spanning domain to anchor the polypeptide or are modified suchthat the membrane spanning domain is non-functional. As used hereinsoluble LFA-3 polypeptides include full-length or truncated (e.g., withinternal deletions) PI-linked LFA-3.

[0038] As used herein, an “antibody homolog” is a protein comprising oneor more polypeptides selected from immunoglobulin light chains,immunoglobulin heavy chains and antigen-binding fragments thereof whichare capable of binding to one or more antigens. The componentpolypeptides of an antibody homolog composed of more than onepolypeptide may optionally be disulfide-bound or otherwise covalentlycrosslinked. Accordingly, antibody homologs include intactimnmunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypesthereof), wherein the light chains of the immunoglobulin may be of typeskappa or lambda. Antibody homologs also include portions of intactimmunoglobulins that retain antigen-binding specificity, for example,Fab fragments, Fab′ fragments, F(ab′)₂ fragments, F(v) fragments, heavychain monomers or dimers, light chain monomers or dimers, dimersconsisting of one heavy and one light chain, and the like.

[0039] As used herein, a “humanized recombinant antibody homolog” is anantibody homolog, produced by recombinant DNA technology, in which someor all of the amino acids of a human immunoglobulin light or heavy chainthat are not required for antigen binding have been substituted for thecorresponding amino acids from a nonhuman mammalian immunoglobulin lightor heavy chain.

[0040] As used herein, a “chimeric recombinant antibody homolog” is anantibody homolog, produced by recombinant DNA technology, in which allor part of the hinge and constant regions of an immunoglobulin lightchain, heavy chain, or both, have been substituted for the correspondingregions from another immunoglobulin light chain or heavy chain.

[0041] Skin Conditions

[0042] The methods of this invention are useful to prevent or treatmammalian, including human, skin conditions characterized by increased Tcell activation and abnormal antigen presentation in the dermis andepidermis, by administering inhibitors of the CD2/LFA-3 interaction.Such conditions include psoriasis, UV damage, atopic dermatitis,cutaneous T cell lymphoma such as mycosis fungoides, allergic andirritant contact dermatitis, lichen planus, alopecia areata, pyodermagangrenosum, vitiligo, ocular cicatricial pemphigoid, and urticaria. Itis to be understood that methods of treatment and prophylaxis of skinconditions such as pyoderma gangrenosum and urticaria are includedwithin the scope of the present invention. These latter skin conditionsare also cyclosporin A sensitive dermatoses and therefore involve T cellactivation. Preferably, the methods of the invention are used in theprophylaxis or treatment of psoriasis or UV damage. The methods of theinvention may be practiced on any mammal, preferably on humans.

[0043] While not wishing to be bound by theory, applicants believe thatinhibitors of the CD2/LFA-3 interaction used in accordance with themethods of this invention are prophylactic and therapeutic for thetreatment of the aforementioned skin conditions because they inhibit theinteraction between T cells and antigen presenting cells, resulting in,among other things, an inhibition of T cell proliferation andactivation. Applicants believe that adverse effects of skin conditionsof the type discussed herein are due to such T cell proliferation andactivation. Applicants believe that the methods of the present inventionare superior to previously available therapies for these skin conditionsfor a number of reasons, including, inhibition of antigen specificinteractions for all antigens present, inhibition of T cell activation,no general immunosuppression and, possibly, induction of tolerance.

[0044] In particular, applicants believe that use of the methods of thisinvention will result in more specific targeting of therapy to T cellsactually in the initiating stage of the lesion with no effect onpolymorphonuclear leukocytes or macrophage mediated effector mechanisms.Accordingly, the patient will be less susceptible to infections thanwith steroids or other general immunosuppressants. Thus, methods ofinhibiting T cell activation, as provided herein, are prophylactic andtherapeutic for such skin conditions.

[0045] Inhibitors Of The CD2/LFA-3 Interaction

[0046] Any inhibitor of the CD2/LFA-3 interaction is useful in themethods of this invention. Such inhibitors include anti-LFA-3 antibodyhomologs, anti-CD2 antibody homologs, soluble LFA-3 polypeptides,soluble CD2 polypeptides, small molecules, e.g., carbohydrates, LFA-3and CD2 mimetic agents and derivatives thereof. Preferred inhibitors aresoluble LFA-3 polypeptides and anti-LFA-3 antibody homologs.

[0047] The utility in the methods of this invention of specific solubleCD2 polypeptides, soluble LFA-3 polypeptides, anti-LFA-3 antibodyhomologs, anti-CD2 antibody homologs or CD2 and LFA-3 mimetic agents mayeasily be determined by assaying their ability to inhibit the LFA-3/CD2interaction. This ability may be assayed, for example, using a simplecell binding assay that permits visual (under magnification) evaluationof the ability of the putative inhibitor to inhibit the interactionbetween LFA-3 and CD2 on cells bearing these molecules. Jurkat cells arepreferred as the CD2⁺ substrate and sheep red blood cells or human JYcells are preferred as the LFA-3⁺ substrate. The binding characteristicsof soluble polypeptides, antibody homologs and mimetic agents useful inthis invention may be assayed in several known ways, such as byradiolabeling the antibody homolog, polypeptide or agent (e.g., ³⁵S or¹²⁵I) and then contacting the labeled polypeptide, mimetic agent orantibody homolog with CD2⁺ of LFA-3⁺ cells, as appropriate. Bindingcharacteristics may also be assayed using an appropriate enzymaticallylabelled secondary antibody. Rosetting competition assays such as thosedescribed by Seed et al. (Proc. Natl. Acad. Sci. USA, 84, pp. 3365-69(1987)) may also be used.

[0048] A. Anti-LFA-3 And Anti-CD2 Antibody Homologs

[0049] Many types of anti-LFA-3 or anti-CD2 antibody homologs are usefulin the methods of this invention. These include monoclonal antibodies,recombinant antibodies, chimeric recombinant antibodies, humanizedrecombinant antibodies, as well as antigen-binding portions of theforegoing.

[0050] Among the anti-LFA-3 antibody homologs, it is preferable to usemonoclonal anti-LFA-3 antibodies. It is more preferable to use amonoclonal anti-LFA-3 antibody produced by a hybridoma selected from thegroup of hybridomas having Accession Nos. ATCC HB 10693 (1E6), ATCC HB10694 (HC-1B11), ATCC HB 10695 (7A6), and ATCC HB 10696 (8B8), or themonoclonal antibody known as TS2/9 (Sanchez-Madrid et al., “ThreeDistinct Antigens Associated with Human T-Lymphocyte-Mediated Cytolysis:LFA-1, LFA-2 and LFA-3”, Proc. Natl. Acad. Sci. USA, 79, pp. 7489-93(1982)). Most preferably, the monoclonal anti-LFA-3 antibody is producedby a hybridoma selected from the group of hybridomas having AccessionNos. ATCC HB 10695 (7A6) and ATCC HB 10693 (1E6).

[0051] Among the anti-CD2 antibody homologs, it is preferable to usemonoclonal anti-CD2 antibodies, such as the anti-CD2 monoclonalantibodies known as the T11₁ epitope antibodies, including TS2/18(Sanchez-Madrid et al., “Three Distinct Antigens Associated with HumanT-Lymphocyte-Mediated Cytolysis: LFA-1, LFA-2 and LFA-3”, Proc. Natl.Acad. Sci. USA, 79, pp. 7489-93 (1982)).

[0052] The technology for producing monoclonal antibodies is well known.Briefly, an immortal cell line (typically myeloma cells) is fused tolymphocytes (typically splenocytes) from a mammal immunized withpreparation comprising a given antigen, and the culture supernatants ofthe resulting hybridoma cells are screened for antibodies against theantigen. See generally, Kohler et al., Nature, “Continuous Cultures ofFused Cells Secreting Antibody of Predefined Specificity”, 256, pp.495-97 (1975). Useful immunogens for the purpose of this inventioninclude CD2- or LFA-3-bearing cells, as well as cell free preparationscontaining LFA-3, CD2 or counter receptor-binding fragments thereof(e.g., CD2 fragments that bind to LFA-3 or LFA-3 fragments that bind toCD2).

[0053] Immunization may be accomplished using standard procedures. Theunit dose and immunization regimen depend on the species of mammalimmunized, its immune status, the body weight of the mammal, etc.Typically, the immunized mammals are bled and the serum from each bloodsample is assayed for particular antibodies using -appropriate screeningassays. For example, useful anti-LFA-3 or anti-CD2 antibodies may beidentified by testing the ability of the immune serum to block sheep redblood cell rosetting of Jurkat cells, which results from the presence ofLFA-3 and CD2 on the respective surfaces of these cells. The lymphocytesused in the production of hybridoma cells typically are isolated fromimmunized mammals whose sera have already tested positive for thepresence of the desired antibodies using such screening assays.

[0054] Typically, the immortal cell line (e.g., a myeloma cell line) isderived from the same mammalian species as the lymphocytes. Preferredimmortal cell lines are mouse myeloma cell lines that are sensitive toculture medium containing hypoxanthine, aminopterin and thymidine (“HATmedium”).

[0055] Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”) 3350. Hybridoma cellsresulting from the fusion are then selected using HAT medium, whichkills unfused and unproductively fused myeloma cells (unfusedsplenocytes die after several days because they are not transformed).Hybridomas producing a desired antibody are detected by screening thehybridoma culture supernatants, for example, for the ability to bind totheir respective counter receptor, or for their ability to block Jurkatcell adhesion to sheep red blood cells. Subcloning of the hybridomacultures by limiting dilution is typically performed to ensuremonoclonality.

[0056] To produce anti-LFA-3 or anti-CD2 monoclonal antibodies,hybridoma cells that tested positive in such screening assays arecultured in a nutrient medium under conditions and for a time sufficientto allow the hybridoma cells to secrete the monoclonal antibodies intothe culture medium. Tissue culture techniques and culture media suitablefor hybridoma cells are well known. The conditioned hybridoma culturesupernatant may be collected and the desired antibodies optionallyfurther purified by well-known methods.

[0057] Alternatively, the desired antibody may be produced by injectingthe hybridoma cells into the peritoneal cavity of a pristane-primedmouse. The hybridoma cells proliferate in the peritoneal cavity,secreting the antibody, which accumulates as ascites fluid. The antibodymay be harvested by withdrawing the ascites fluid from the peritonealcavity with a syringe.

[0058] Anti-CD2and anti-LFA-3 antibody homologs useful in the presentinvention may also be recombinant antibodies produced by host cellstransformed with DNA encoding immunoglobulin light and heavy chains of adesired antibody. Recombinant antibodies may be produced by well knowngenetic engineering techniques. See, e.g., U.S. Pat. No. 4,816,397,which is incorporated herein by reference.

[0059] For example, recombinant antibodies may be produced by cloningcDNA or genomic DNA encoding the immunoglobulin light and heavy chainsof the desired antibody from a hybridoma cell that produces an antibodyhomolog useful in this invention. The cDNA or genomic DNA encoding thosepolypeptides is then inserted into expression vectors so that both genesare operatively linked to their own transcriptional and translationalexpression control sequences. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. Typically, both genes are inserted into the same expressionvector.

[0060] Prokaryotic or eukaryotic host cells may be used. Expression ineukaryotic host cells is preferred because such cells are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. However, any antibody produced that isinactive due to improper folding may be renaturable according to wellknown methods (Kim and Baldwin, “Specific Intermediates in the FoldingReactions of Small Proteins and the Mechanism of Protein Folding”, Ann.Rev. Biochem., 51, pp. 459-89 (1982)). It is possible that the hostcells will produce portions of intact antibodies, such as light chaindimers or heavy chain dimers, which also are antibody homologs accordingto the present invention.

[0061] It will be understood that variations on the above procedure areuseful in the present invention. For example, it may be desired totransform a host cell with DNA encoding either the light chain or theheavy chain (but not both) of an antibody homolog. Recombinant DNAtechnology may also be used to remove some or all of the DNA encodingeither or both of the light and heavy chains that is not necessary forCD2 or LFA-3 counter receptor binding. The molecules expressed from suchtruncated DNA molecules are useful in the methods of this invention. Inaddition, bifunctional antibodies may be produced in which one heavy andone light chain are anti-CD2 or anti-LFA-3 antibody homologs and theother heavy and light chain are specific for an antigen other than CD2or LFA-3, or another epitope of CD2 or LFA-3.

[0062] Chimeric recombinant anti-LFA-3 or anti-CD2 antibody homologs maybe produced by transforming a host cell with a suitable expressionvector comprising DNA encoding the desired immunoglobulin light andheavy chains in which all or some of the DNA encoding the hinge andconstant regions of the heavy and/or the light chain have beensubstituted with DNA from the corresponding region of an immunoglobulinlight or heavy chain of a different species. When the originalrecombinant antibody is nonhuman, and the inhibitor is to beadministered to a human, substitution of corresponding human sequencesis preferred. An exemplary chimeric recombinant antibody has mousevariable regions and human hinge and constant regions. See generally,U.S. Pat. No. 4,816,397 and Morrison et al., “Chimeric Human AntibodyMolecules: Mouse Antigen-Binding Domains With Human Constant RegionDomains”, Proc. Natl. Acad. Sci. USA, 81, pp. 6851-55 (1984).

[0063] Humanized recombinant anti-LFA-3 or anti-CD2 antibodies may beproduced by transforming a host cell with a suitable expression vectorcomprising DNA encoding the desired nonhuman immunoglobulin light andheavy chains in which all or some of the DNA encoding amino acids notinvolved in antigen binding-have been substituted with DNA from thecorresponding region of a desired human immunoglobulin light or heavychain. See generally, Jones et al., “Replacing theComplementarity-Determining Regions in a Human Antibody with Those froma Mouse”, Nature, 321, pp.522-25 (1986).

[0064] Anti-CD2 and anti-LFA-3 antibody homologs that are not intactantibodies are also useful in this invention. Such homologs may bederived from any of the antibody homologs described above. For example,antigen-binding fragments, as well as full-length monomeric, dimeric ortrimeric polypeptides derived from the above-described antibodies arethemselves useful. Useful antibody homologs of this type include Fabfragments, Fab′ fragments, F(ab′)₂ fragments, F(v) fragments, heavychain monomers or dimers, light chain monomers or dimers, dimersconsisting of one heavy and one light chain, and the like. Anti-LFA-3heavy chains are preferred anti-LFA-3 antibody fragments.

[0065] Antibody fragments may also be produced by chemical methods,e.g., by cleaving an intact antibody with a protease, such as pepsin orpapain, and optionally treating the cleaved product with a reducingagent. Alternatively, useful fragments may be produced by using hostcells transformed with truncated heavy and/or light chain genes. Heavyand light chain monomers may be produced by treating an intact antibodywith a reducing agent, such as dithiothreitol, followed by purificationto separate the chains. Heavy and light chain monomers may also beproduced by host cells transformed with DNA encoding either the desiredheavy chain or light chain, but not both. See, e.g., Ward et al.,“Binding Activities of a Repertoire of Single Immunoglobulin VariableDomains Secreted from Escherichia coli”, Nature, 341, pp. 544-46 (1989);Sastry et al., “Cloning of the Immunological Repertoire in Escherichiacoli for Generation of Monoclonal Catalytic Antibodies: Construction ofa Heavy Chain Variable Region-Specific cDNA Library”, Proc. Natl. Acad.Sci. USA, 86, pp. 5728-32 (1989).

[0066] B. Soluble CD2 and LFA-3 Polypeptides

[0067] Soluble LFA-3 polypeptides or soluble CD2 polypeptides thatinhibit the interaction of LFA-3 and CD2 are useful in the methods ofthe present invention. Soluble LFA-3 polypeptides are preferred.

[0068] Soluble LFA-3 polypeptides may be derived from the transmembraneform of LFA-3, particularly the extracellular domain (e.g., AA₁-AA₁₈₇ ofSEQ ID NO:2). Such polypeptides are described in U.S. Pat. No. 4,956,281and co-pending U.S. patent application Ser. No. 07/667,971 (which sharesa common assignee with the present application), which are hereinincorporated by reference. Preferred soluble LFA-3 polypeptides includepolypeptides consisting of AA₁-AA₉₂ of SEQ ID NO:2, AA₁-AA₈₀ of SEQ IDNO:2, AA₅₀-AA₆₅ of SEQ ID NO:2 and AA₂₀-AA₈₀ of SEQ ID NO:2. A vectorcomprising a DNA sequence encoding SEQ ID NO:2 (i.e., SEQ ID NO:1) isdeposited with the American Type Culture Collection, Rockville, Md.under Accession No. 75107.

[0069] The most preferred fusion proteins of this type contain the aminoterminal 92 amino acids of mature LFA-3, the C-terminal 10 amino acidsof a human IgG1 hinge region containing the two cysteine residuesthought to participate in interchain disulfide bonding, and the C_(H)2and C_(H)3 regions of a human IgG₁ heavy chain constant domain (e.g.,SEQ ID NO:8). This fusion protein is referred to herein as “LFA3TIP.” Aplasmid, pSAB152, encoding an exemplary LFA3TIP is deposited withAmerican Type Culture Collection, Rockville, Md., under the accessionnumber ATCC 68720. The DNA sequence of the pSAB152 insert is SEQ IDNO:7.

[0070] One way of producing LFA3TIP for use in the methods of thisinvention is described in co-pending, commonly assigned U.S. patentapplication Ser. No. 07/770,967. Generally, conditioned culture mediumof COS7 or CHO cells transfected with pSAB152 was concentrated using anAMICON S1Y30 spiral cartridge system (AMICON, Danvers, Mass.) andsubjected to Protein A-Sepharose 4B (Sigma, St. Louis, Mo.)chromatography. The bound proteins were eluted and subjected toSuperose-12 (Pharmacia/LKB, Piscataway, N.J.) gel filtrationchromatography.

[0071] Superose-12 fractions containing LFA3TIP with the least amount ofcontaminating proteins, as determined on SDS-PAGE gels and by Westernblot analysis, (see, e.g., Towbin et al., Proc. Natl. Acad. Sci. USA,74, pp. 4350-54 (1979); Antibodies: A Laboratory Manual, pp. 474-510(Cold Spring Harbor Laboratory (1988)), were pooled and concentrated ina YM30 Centricon (AMICON). LFA3TIP was detected on Western blots using arabbit anti-LFA-3 polyclonal antiserum, followed by detectably labeledgoat anti-rabbit IgG. The purified LFA3TIP of COS7 or CHO cells was adimer of two monomeric LFA-3-Ig fusion proteins, connected by disulfidebonds.

[0072] Another preferred fusion protein consists of the first and secondLFA-3 domain fused to the hinge C_(H)2 and C_(H)3 regions of human IgG1,herein referred to as LLFA3-Ig.

[0073] Soluble LFA-3 polypeptides may also be derived from the PI-linkedform of LFA-3, such as those described in PCT Patent Application SerialNo. WO 90/02181. A vector comprising a DNA sequence encoding PI-linkedLFA-3 (i.e., SEQ ID NO:3) is deposited with the American Type CultureCollection, Rockville, Md. under Accession No. 68788. It is to beunderstood that the PI-linked form of LFA-3 and the transmembrane formof LFA-3 have identical amino acid sequences through the entireextracellular domain. Accordingly, the preferred PI-linked LFA-3polypeptides are the same as for the transmembrane form of LFA-3.

[0074] Soluble CD2 polypeptides may be derived from full length CD2,particularly the extracellular domain (e.g., AA₁-AA₁₈₅ of SEQ ID NO:6).Such polypeptides may comprise all or part of the extracellular domainof CD2. Exemplary soluble CD2 polypeptides are described in PCT WO90/08187, which is herein incorporated by reference.

[0075] The production of the soluble polypeptides useful in thisinvention may be achieved by a variety of methods known in the art. Forexample, the polypeptides may be derived from intact transmembrane LFA-3or CD2 molecules or an intact PI-linked LFA-3 molecule by proteolysisusing specific endopeptidases in combination with exopeptidases, Edmandegradation, or both. The intact LFA-3 molecule or the intact CD2molecule, in turn, may be purified from its natural source usingconventional methods. Alternatively, the intact LFA-3 or CD2 may beproduced by known recombinant DNA techniques using cDNAs (see, e.g.,U.S. Pat. No. 4,956,281 to Wallner et al.; Aruffo and Seed, Proc. Natl.Acad. Sci., 84, pp. 2941-45 (1987); Sayre et al., Proc. Natl. Acad. Sci.USA, 84, pp. 2941-45 (1987)).

[0076] Preferably, the soluble polypeptides useful in the presentinvention are produced directly, thus eliminating the need for an entireLFA-3 molecule or an entire CD2 molecule as a starting material. Thismay be achieved by conventional chemical synthesis techniques or bywell-known recombinant DNA techniques wherein only those DNA sequenceswhich encode the desired peptides are expressed in transformed hosts.For example, a gene which encodes the desired soluble LFA-3 polypeptideor soluble CD2 polypeptide may be synthesized by chemical means using anoligonucleotide synthesizer. Such oligonucleotides are designed based onthe amino acid sequence of the desired soluble LFA-3 polypeptide orsoluble CD2 polypeptide. Specific DNA sequences coding for the desiredpeptide also can be derived from the full length DNA sequence byisolation of specific restriction endonuclease fragments or by PCRsynthesis of the specified region.

[0077] Standard methods may be applied to synthesize a gene encoding asoluble LFA-3 polypeptide or a soluble CD2 polypeptide that is useful inthis invention. For example, the complete amino acid sequence may beused to construct a back-translated gene. A DNA oligomer containing anucleotide sequence coding for a soluble LFA-3 polypeptide or a solubleCD2 polypeptide useful in this invention may be synthesized in a singlestep. Alternatively, several smaller oligonucleotides coding forportions of the desired polypeptide may be synthesized and then ligated.Preferably, a soluble LFA-3 polypeptide or a soluble CD2 polypeptideuseful in this invention will be synthesized as several separateoligonucleotides which are subsequently linked together. The individualoligonucleotides typically contain 5′ or 3′ overhangs for complementaryassembly.

[0078] Once assembled, preferred genes will be characterized bysequences that are recognized by restriction endonucleases (includingunique restriction sites for direct assembly into a cloning or anexpression vector), preferred codons taking into consideration the hostexpression system to be used, and a sequence which, when transcribed,produces a stable, efficiently translated mRNA. Proper assembly may beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host.

[0079] It will be appreciated by those of skill in the art that, due tothe degeneracy of the genetic code, DNA molecules comprising many othernucleotide sequences will also be capable of encoding the soluble LFA-3and CD2 polypeptides encoded by the specific DNA sequences describedabove. These degenerate sequences also code for polypeptides that areuseful in this invention.

[0080] The DNA sequences may be expressed in unicellular hosts. As iswell known in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host. Preferably, the expressioncontrol sequences, and the gene of interest, will be contained in anexpression vector that further comprises a bacterial selection markerand origin of replication. If the expression host is a eukaryotic cell,the expression vector should further comprise an additional expressionmarker useful in the expression host.

[0081] The DNA sequences encoding the desired soluble polypeptides mayor may not encode a signal sequence. If the expression host isprokaryotic, it generally is preferred that the DNA sequence not encodea signal sequence. If the expression host is eukaryotic, it generally ispreferred that a signal sequence be encoded.

[0082] An amino terminal methionine may or may not be present on theexpressed product. If the terminal methionine is not cleaved by theexpression host, it may, if desired, be chemically removed by standardtechniques.

[0083] A wide variety of expression host/vector combinations may beemployed. Useful expression vectors for eukaryotic hosts, include, forexample, vectors comprising expression control sequences from SV40,bovine papilloma virus, adenovirus and cytomegalovirus. Usefulexpression vectors for bacterial hosts include known bacterial plasmids,such as plasmids from E. coli, including col E1, pCR1, pBR322, pMB9 andtheir derivatives, wider host range plasmids, such as RP4, phage DNAs,e.g., the numerous derivatives of phage lambda, e.g., NM989, and otherDNA phages, such as M13 and filamentous single stranded DNA phages.Useful expression vectors for yeast cells include the 2μ plasmid andderivatives thereof. Useful vectors for insect cells include pVL 941.

[0084] In addition, any of a wide variety of expression controlsequences may be used in these vectors. Such useful expression controlsequences include the expression control sequences associated withstructural genes of the foregoing expression vectors. Examples of usefulexpression control sequences include, for example, the early and latepromoters of SV40 or adenovirus, the lac system, the trp system, the TACor TRC system, the major operator and promoter regions of phage lambda,the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., Pho5, the promoters of the yeast α-mating systemand other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof.

[0085] A wide variety of unicellular host cells are useful. These hostsmay include well known eukaryotic and prokaryotic hosts, such as strainsof E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insectcells such as Spodoptera frugiperda (SF9), animal cells such as CHO andmouse cells, African green monkey cells such as COS 1, COS 7, BSC 1, BSC40, and BMT 10, and human cells, as well as plant cells in tissueculture. For animal cell expression, we prefer CHO cells and COS 7cells.

[0086] It should, of course, be understood that not all vectors andexpression control sequences will function equally well to express theDNA sequences described herein. Neither will all hosts function equallywell with the same expression system. However, one of skill in the artmay make a selection among these vectors, expression control sequencesand hosts without undue experimentation. For example, in selecting avector, the host must be considered because the vector must replicate init. The vector's copy number, the ability to control that copy number,and the expression of any other proteins encoded by the vector, such asantibiotic markers, should also be considered.

[0087] In selecting an expression control sequence, a variety of factorsshould also be considered. These include, for example, the relativestrength of the sequence, its controllability, and its compatibilitywith the DNA sequences discussed herein, particularly as regardspotential secondary structures. Unicellular hosts should be selected byconsideration of their compatibility with the chosen vector, thetoxicity of the product coded for by the DNA sequences, their secretioncharacteristics, their ability to fold the soluble polypeptidescorrectly, their fermentation or culture requirements, and the ease ofpurification of the products coded for by the DNA sequences.

[0088] Within these parameters, one of skill in the art may selectvarious vector/expression control sequence/host combinations that willexpress the desired DNA sequences on fermentation or in large scaleanimal culture, for example with CHO cells or COS 7 cells.

[0089] The soluble LFA-3 and CD2 polypeptides may be isolated from thefermentation or cell culture and purified using any of a variety ofconventional methods. One of skill in the art may select the mostappropriate isolation and purification techniques.

[0090] While recombinant DNA techniques are the preferred method ofproducing useful soluble CD2 polypeptides or soluble LFA-3 polypeptideshaving a sequence of more than 20 amino acids, shorter CD2 or LFA-3polypeptides having less than about 20 amino acids are preferablyproduced by conventional chemical synthesis techniques. Syntheticallyproduced polypeptides useful in this invention can advantageously beproduced in extremely high yields and can be easily purified.

[0091] Preferably, such soluble CD2 polypeptides or soluble LFA-3polypeptides are synthesized by solution phase or solid phasepolypeptide synthesis and, optionally, digested with carboxypeptidase(to remove C-terminal amino acids) or degraded by manual Edmandegradation (to remove N-terminal amino acids). Proper folding of thepolypeptides may be achieved under oxidative conditions which favordisulfide bridge formation as described by Kent, “Chemical Synthesis ofPolypeptides and Proteins”, Ann. Rev. Biochem., 57, pp. 957-89 (1988).Polypeptides produced in this way may then be purified by separationtechniques widely known in the art, preferably utilizing reverse phaseHPLC. The use of solution phase synthesis advantageously allows for thedirect addition of certain derivatized amino acids to the growingpolypeptide chain, such as the O-sulfate ester of tyrosine. Thisobviates the need for a subsequent derivatization step to modify anyresidue of the polypeptides useful in this invention.

[0092] C. LFA-3 And CD2 Mimetic Agents

[0093] Also useful in the methods of this invention are LFA-3 and CD2mimetic agents. These agents which may be peptides, semi-peptidiccompounds or non-peptidic compounds, are inhibitors of the CD2/LFA-3interaction. The most preferred CD2 and LFA-3 mimetic agents willinhibit the CD2/LFA-3 interaction at least as well as anti-LFA-3monoclonal antibody 7A6 or anti-CD2 monoclonal antibody TS2/18(described supra).

[0094] Such mimetic agents may be produced by synthesizing a pluralityof peptides (e.g., 5-20 amino acids in length), semi-peptidic compoundsor non-peptidic, organic compounds, and then screening those compoundsfor their ability to inhibit the CD2/LFA-3 interaction. See generallyU.S. Pat. No. 4,833,092, Scott and Smith, “Searching for Peptide Ligandswith an Epitope Library”, Science, 249, pp. 386-90 (1990), and Devlin etal., “Random Peptide Libraries: A Source of Specific Protein BindingMolecules”, Science, 249, pp. 404-07 (1990), which are hereinincorporated by reference.

[0095] D. Derivatized Inhibitors

[0096] Also useful in the methods of this invention are derivatizedinhibitors of the CD2/LFA-3 interaction in which, for example, any ofthe antibody homologs, soluble CD2 and LFA-3 polypeptides, or CD2 andLFA-3 mimetic agents described herein are functionally linked (bychemical coupling, genetic fusion or otherwise) to one or more membersindependently selected from the group consisting of anti-LFA-3 andanti-CD2 antibody homologs, soluble LFA-3 and CD2 polypeptides, CD2 andLFA-3 mimetic agents, cytotoxic agents and pharmaceutical agents.

[0097] One type of derivatized inhibitor is produced by crosslinking twoor more inhibitors (of the same type or of different types). Suitablecrosslinkers include those that are heterobifunctional, having twodistinctly reactive groups separated by an appropriate spacer (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional(e.g., disuccinimidyl suberate). Such linkers are available from PierceChemical Company, Rockford, Ill.

[0098] Another possibility for cross-linking takes advantage of the PIlinkage signal sequence in PI-linked LFA-3, or fragments thereof.Specifically, DNA encoding the PI-linkage signal sequence (e.g.,AA₁₆₂-AA₂₁₂ of SEQ ID NO:4) is ligated downstream of DNA encoding adesired polypeptide, preferably a soluble LFA-3 polypeptide. If thisconstruct is expressed in an appropriate eukaryotic cell, the cell willrecognize the PI linkage signal sequence and will covalently link PI tothe polypeptide. The hydrophobic property of the PI may then beexploited to form micellar aggregates of the polypeptides.

[0099] Also useful are inhibitors linked to one or more cytotoxic orpharmaceutical agents. Useful pharmaceutical agents include biologicallyactive peptides, polypeptides and proteins, such as antibody homologsspecific for a human polypeptide other than CD2 or LFA-3, or portionsthereof. Useful pharmaceutical agents and cytotoxic agents also includecyclosporin A, prednisone, FK506, methotrexate, steroids, retinoids,interferon, and nitrogen mustard.

[0100] Preferred inhibitors derivatized with a pharmaceutical agentinclude recombinantly-produced polypeptides in which a soluble LFA-3polypeptide, soluble CD2 polypeptide, or a peptidyl CD2 or peptidylLFA-3 mimetic agent is fused to all or part of an immunoglobulin heavychain hinge region and all or part of a heavy chain constant region.Preferred-polypeptides for preparing such fusion proteins are solubleLFA-3 polypeptides. Most preferred are fusion proteins containingAA₁-AA₉₂ of LFA-3 (e.g., SEQ ID NO:2) fused to a portion of a human IgG₁hinge region (including the C-terminal ten amino acids of the hingeregion containing two cysteine residues thought to participate ininterchain disulfide bonding) and the C_(H)2 and C_(H)3 regions of anIgG₁ heavy chain constant domain. Such fusion proteins are expected toexhibit prolonged serum half-lives and enable inhibitor dimerization.

[0101] Pharmaceutical Compositions And Methods According To ThisInvention

[0102] This invention provides a method for preventing or treating theabove-mentioned skin conditions in a mammal by administering to themammal one or more inhibitors of the CD2/LFA-3 interaction, orderivatized form(s) thereof.

[0103] Preferably, an effective amount of the inhibitor or derivatizedform thereof is administered. By “effective amount” is meant an amountcapable of lessening the spread or severity of the skin conditionsdescribed herein.

[0104] It will be apparent to those of skill in the art that theeffective amount of inhibitor will depend, inter alia, upon theadministration schedule, the unit dose administered, whether theinhibitor is administered in combination with other therapeutic agents,the immune status and health of the patient, the therapeutic orprophylactic activity of the particular inhibitor administered and theserum half-life.

[0105] Preferably, the inhibitor is administered at a dose between about0.001 and about 50 mg inhibitor per kg body weight, more preferably,between about 0.01 and about 10 mg inhibitor per kg body weight, mostpreferably between about 0.1 and about 4 mg inhibitor per kg bodyweight.

[0106] Unit doses should be administered until an effect is observed.The effect may be measured by a variety of methods, including, in vitroT cell activity assays and clearing of affected skin areas. Preferably,the unit dose is administered about one to three times per week or oneto three times per day. More preferably, it is administered about one tothree times per day for between about 3 and 7 days, or about one tothree times per day for between about 3 and 7 days on a monthly basis.It will be recognized, however, that lower or higher dosages and otheradministrations schedules may be employed.

[0107] The inhibitor(s) or derivatized form(s) thereof are alsopreferably administered in a composition including a pharmaceuticallyacceptable carrier. By “pharmaceutically acceptable carrier” is meant acarrier that does not cause an allergic reaction or other untowardeffect in patients to whom it is administered.

[0108] Suitable pharmaceutically acceptable carriers include, forexample, one or more of water, saline, phosphate buffered saline,dextrose, glycerol, ethanol and the like, as well as combinationsthereof. Pharmaceutically acceptable carriers may further comprise minoramounts of auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the inhibitor.

[0109] The pharmaceutical composition or inhibitor may be administeredin conjunction with other therapeutic or prophylactic agents. Theseinclude, for example, cyclosporin A, steroids, retinoids, nitrogenmustard, interferon, methotrexate, antibiotics and antihistamines.

[0110] These agents may be administered in single dosage form with theinhibitor (i.e., as part of the same pharmaceutical composition), amultiple dosage form separately from the inhibitor, but concurrently, ora multiple dosage form wherein the two components are administeredseparately but sequentially. Alternatively, the inhibitor and the otheractive agent may be in the form of a single conjugated molecule.Conjugation of the two components may be achieved by standardcross-linking techniques well known in the art. A single molecule mayalso take the form of a recombinant fusion protein. In addition, theinhibitors, or pharmaceutical compositions, useful in the presentinvention may be used in combination with other therapies such as PUVA,chemotherapy and UV light. Such combination therapies may advantageouslyutilize lower dosages of the therapeutic or prophylactic agents.

[0111] The inhibitor, or pharmaceutical composition, may be in a varietyof forms. These include, for example, solid, semi-solid and liquiddosage forms, such as tablets, pills, powders, liquid solutions,dispersions or suspensions, liposomes, suppositories, injectableinfusible, and topical preparations. The preferred form depends on theintended mode of administration and therapeutic application. Thepreferred forms are injectable or infusible solutions.

[0112] The inhibitor or pharmaceutical composition may be administeredintravenously, intramuscularly, subcutaneously, intra-articularly,intrathecally, periostally, intratumorally, intralesionally,perilesionally by infusion, orally, topically or by inhalation.Preferably it is administered subcutaneously, intramuscularly orintravenously. Most preferably, it is administered subcutaneously.

[0113] The invention includes formulations suitable for use as topicallyapplied sunscreens or UV-protectants. Preferred embodiments includeLFA3TIP preparations. The active ingredient can be formulated in aliposome. The product can be applied before, during, or after UVexposure, or before, during, or after the development of redness.

[0114] In order that this invention may be better understood, thefollowing examples are set forth These examples are for purposes ofillustration only, and are not to be construed as limiting the scope ofthe invention in any manner.

EXAMPLE 1

[0115] Subjects

[0116] Six adult patients participated in the investigation. Informedconsent was obtained after Internal Review Board approval of theprotocol. All patients satisfied the major diagnostic criteria forpsoriasis, namely chronic papulosquamous plaques of characteristicmorphology and distribution. The intermittent use of topicalcorticosteroids was common among these patients but was discontinued 2weeks prior to entry into the study. A group of healthy volunteers withno history of psoriasis or other skin disease was utilized as the normalcontrol group.

[0117] Preparation of Epidermal Cell Suspensions

[0118] Skin biopsy specimens were obtained from both normal and lesionalskin by using a keratome. The specimens were submerged in Dulbecco'sphosphate buffered saline (“PBS”) (Gibco Labs, Grand Island, N.Y.)containing 50 units/ml dispase (Collaborative Research, Bedford, Mass.).The-specimens were then incubated at 4° C. for 18 hours and theepidermis removed from the remaining dermis.

[0119] Epidermal sheets were removed from the dermis, submerged inDulbecco's PBS containing 0.5% trypsin (Sigma Chemical Co., St. Louis,Mo.), and incubated at 37° C. for 30 minutes.

[0120] Trypsinized epidermal sheets were transferred to 0.05% DNase(Sigma) in Dulbecco's PBS where they were teased into a cell suspension.Fetal bovine serum (“FBS”) (Hyclone, Logan, Utah) was added toinactivate residual trypsin and the epidermal cell suspension thenpassed through a 112 μm nylon filter (Tetko, Elmsford, N.Y.). Afterwashing the predominantly single cell suspension three times inDulbecco's PBS with 1% FBS, cells were resuspended in culture mediawhich consisted of RPMI 1640 (Whittaker, Mass.; Bioproducts, Wakerfield,Md.) containing 1% penicillin and streptomycin, 1% glutamine (Gibco),and 10% human AB serum (Sigma).

[0121] Preparation of MNC and T cells

[0122] Peripheral blood mononuclear cells (“MNC”) were isolated fromheparinized blood using Ficoll-Hypaque (Pharmacia) density gradientcentrifugation according to manufacturer's suggested protocol. RestingCD4+ T cells were prepared as follows. Macrophages were removed byplastic adherence at 37° C. for 1 hour. The nonadherent,macrophage-depleted MNC were washed, and then depleted of CD8⁺ Tlymphocytes, activated T cells, B cells, antigen presenting cells and NKcells by incubation with monoclonal antibodies to CD8 (ATCC CRL 8014),HLA-DR (ATCC CRL H355), and CD11b (ATCC CRL 8026). These antibodies wereused as dilutions in PBS (1:200) of ascites fluid from pristane-primedmice.

[0123] The antibody treated MNC were incubated at 4° C. with 4.5 nmmagnetic particles coated with goat anti-mouse IgG (Dynabeads M-450,Dynal, Oslo, Norway) at a ratio of 3 beads per cell. Antigen positivecells were depleted by being drawn by a magnet (Advanced Magnetics,Cambridge, Mass.) against the side of the tube allowing the remainingcells in suspension to be decanted. The decanted cell suspension wasagain exposed to a magnet and cells remaining in suspension collected.Fresh goat anti-mouse IgG beads were again added to the collected cellsin suspension in order to deplete any remaining antigen positive cells,and the magnetic removal process repeated. Cells were washed in PBS andresuspended in culture media prior to use. This treatment results in apreparation of resting CD4⁺ T lymphocytes enriched to 99% purity anddevoid of intrinsic antigen presenting activity.

[0124] Proliferative Response of T Lymphocytes to Autologous PsoriaticCells

[0125] One hundred thousand CD4⁺ T lymphocytes were added to roundbottom microtiter wells (Costar, Cambridge, Mass.) with eighty thousandpsoriatic epidermal cells in 0.2 ml of RPMI containing 10% human ABserum (Sigma, St. Louis, Mo.). This number of psoriatic epidermal cellsper well was chosen because previous experiments demonstrated that thisnumber is sufficient to induce autoreactive T cell responses. Afterincubation at 37° C. in 5% CO₂/95% air for 6 days, 1 μCi of [³H]TdR (ICNRadiochemicals, Irvine, Calif.) was added per well and the cellsharvested 18 hours later on a PHD cell harvester (Cambridge TechnologyInc., Cambridge, Mass.). The [³H]TdR incorporation was measured on aPackard scintillation counter (Packard Instrument Co., Downers Grover.Ill.). [³H]TdR incorporation is a measure of T cell proliferation.

[0126] Appropriate controls for T cells (“TC”) alone or epidermal cells(“EC”) alone were carried out using the above protocol. No [³H]TdRincorporation was observed in these assays (data not shown). Briskproliferation of autologous T cells in response to psoriatic skin cellswas observed (data not shown).

[0127] In addition, to test the allogeneic response to normal skin, theabove protocol was carried out using one hundred thousand allogeneic Tcells and eighty thousand normal skin cells. Under these conditions, abrisk proliferation of allogeneic T cells was observed (data not shown).

[0128] Blocking of Psoriatic Epidermal Cells' Ability To StimulateAutologous T Lymphocyte Proliferation

[0129] The effect on [³H]TdR incorporation (i.e., T cell proliferation)of an anti-CD2 monoclonal antibody (TS2/18) (Sanchez-Madrid et al.,“Three Distinct Antigens Associated with Human T-lymphocyte-mediatedCytolysis: LFA-1, LFA-2, and LFA-3”, Proc. Natl. Acad. Sci. USA, 79, pp.7489-93 (1982)), an anti-LFA-3 monoclonal antibody (7A6) (ATCC HB10695), or an isotype-matched, control monoclonal antibody of irrelevantspecificity (MOPC21, Sigma Chemical Co., St. Louis, Mo.) was measuredusing the protocol outlined above in the presence of 50 μg/ml of therespective antibodies.

[0130]FIG. 1 demonstrates that addition of anti-CD2 or anti-LFA-3resulted in a consistent (n=4) and substantial (approximately 60%)inhibition of autologous T cell proliferation in response to lesionalpsoriatic epidermis, as compared to proliferation in the presence of theisotype-matched control antibody.

[0131]FIG. 1 displays data for four patients only. These four patientsdemonstrated autoreactivity of blood CD4⁺ T cells to their own lesionalepidermis, despite the fact that no antigen was added to the system.This is an abnormal finding; normal individuals' cocultures ofautologous blood T cells and epidermal cells do not react. Such areaction is considered to be an in vitro model of autoimmune reactionsoccurring in the skin. EC preparations from two additional patients werenot informative. One EC preparation was bacterially contaminated; theother contained antigen presenting cells that did not induceautoreactive T cell responses.

[0132] Addition of 50 μg per ml of the anti-CD2 or anti-LFA-3 antibodiesto the allogeneic normal skin assay described above also resulted in aninhibition of allogeneic T cell activation. The degree of inhibition wasnot as substantial (approximately 40%) as that observed for autologousantigen presenting cell activity when using lesional psoriatic epidermis(data not shown).

[0133] Addition of the isotype-matched control antibody (specific for anirrelevant antigen) did not significantly alter the level of T cellproliferation of autologous T cells induced by lesional psoriaticepidermis (data not shown).

EXAMPLE 2

[0134] Subject

[0135] One adult subject participated in this investigation. Informedconsent was obtained after Internal Review Board approval of theprotocol. The minimal dose of UV B from a bank of fluorescent bulbs (FS40) required to induce skin erythema in the subject was determined priorto the study. A moderate sunburn (4 minimal erythemal doses) was thenadministered to the left buttock, which 3 days later was the source ofUV damaged skin. Skin from the right buttock, which was unburned, wasutilized for the control.

[0136] Preparation of Epidermal Cell Suspensions

[0137] Skin biopsy specimens were obtained from both normal andsunburned skin by using a keratome. Epidermal cell suspensions wereprepared from these specimens using substantially the same protocol asin Example 1.

[0138] Isolation and Depletion of T cells

[0139] Peripheral blood mononuclear cells (“MNC”) were isolated fromheparinized blood of another person, using Ficoll-Hypaque (Pharmacia)density gradient centrifugation according to manufacturer's suggestedprotocol. CD4⁺ T lymphocytes were then prepared substantially asoutlined in Example 1.

[0140] Proliferative Response Of T Lymphocytes To Allogeneic UV DamagedEpidermal Cells

[0141] One hundred thousand CD4⁺ T lymphocytes from another individualwere added to round bottom microtiter wells (Costar, Cambridge, Mass.)with UV damaged epidermal cells from the subject, incubated in thepresence of [³H]TdR, harvested and [³H]TdR incorporation was measuredsubstantially as outlined in Example 1. This example differs fromExample 1 in that the antigenic stimulus is alloantigen, rather thanautoantigens that are stimulatory in psoriasis. Thus, allogeneic T cellswere used, rather than autologous T cells.

[0142]FIG. 2 shows a brisk proliferation of allogeneic T cells (asmeasured by [³H]TdR incorporation) when incubated with UV damagedepidermal cells (“EC+TC”).

[0143] Blocking Of UV Damaged Epidermal Cells' Ability To StimulateAllogeneic T Lymphocyte Proliferation

[0144] The effect on [³H]TdR incorporation (i.e., T cell proliferation)of an anti-LFA-3 monoclonal antibody (1E6) (ATCC HB 10693), an anti-CD2monoclonal antibody (TS2/18) (Sanchez-Madrid et al., “Three DistinctAntigens Associated With Human T-lymphocyte-Mediated Cytolysis: LFA-1,LFA-2,and LFA-3”, Proc. Natl. Acad. Sci. USA. 79, pp. 7489-93 (1982)),and an isotype-matched, control monoclonal antibody of irrelevantspecificity (MOPC21, Sigma Chemical Co.), was measured using theprotocol outlined above in the presence of 50 μg/ml of the respectiveantibodies.

[0145]FIG. 2 shows that in the presence of a monoclonal antibody ofirrelevant specificity (MOPC21, Sigma Chemical Co.), [³H]TdRincorporation was somewhat reduced. However, the addition of anti-LFA-3monoclonal antibody 1E6 or anti-CD2 monoclonal antibody TS2/18 resultedin a substantial inhibition of T cell proliferation compared toproliferation in the presence of the control antibody.

EXAMPLE 3

[0146] Preparation of LFA3TIP

[0147] LFA3TIP, a fusion protein comprised of the first extracellulardomain of LFA-3 fused to the hinge, C_(H)2 and C_(H)3 regions of humanIgG1 was constructed as described in Miller, G T et al. (1993) J. Exp.Med.178:211, hereby incorporated by-reference. LFA3TIP was purified fromculture medium of transfectant CHO (chinese hamster ovary) cell lines byabsorption to Protein-A Sepharose 4B (Phannacia) and eluted with 50 mMglycine, 250 mM NaCl (pH 3.0). Fractions containing protein were pooledand subjected to gel filtration on Superose-6 (Pharmacia) in phosphatebuffered saline (PBS). Peak fractions were pooled and analyzed forpurity on 12% reducing and non-reducing SDS-PAGE.

[0148] 1. LFA3TIP Results in a Decrease of the Proliferative Response ofAutologous Mononuclear Cells to Lesional Psoriatic Epidermal Cells

[0149] The ability of LFA3TIP to inhibit the response of autologousmononuclear cells (MNC) to lesional psoriatic epidermal cells (EC) insuspension was determined. Epidermal cell suspensions were preparedessentially in the same way as in Example 1 above. Proliferation of MNCwas measured by thymidine incorporation. As shown in FIG. 3 there wasminimal proliferation with epidermal cells alone. Combination of EC withMNC resulted in a relatively strong response, approaching 6000 cpm. Incontrast to the human IgG controls, addition of LFA3TIP, atconcentrations between 5 and 0.3 μg/ml, resulted in a consistentinhibition of the autoreactive response. If the counts of EC and MNCalone are taken into account, the LFA3TIP inhibition is quitesubstantial.

[0150] The mechanism of the IgG1 enhancement of EC-induced MNCproliferation is unknown. It is clear however, from the experimentdescribed above, using autologous MNC and lesional epidermal cells ofpsoriasis, that LFA3TIP results in very substantial inhibition.

[0151] 2. Spontaneous Proliferation of Lesional Psoriatic Dermal Cells

[0152]FIG. 4 shows the effect of LFA3TIP on spontaneous lesionalpsoriatic dermal cell proliferation. Suspensions of psoriatic dermiswere prepared by dispase splitting of the epidermis from the dermis,followed by digestion of the dermis with collagenase, hyalurinidase, andDNase, and filtering through sequential nylon mesh sizing filters.Psoriatic dermis, upon digestion into a single cell suspension,undergoes an increased level of spontaneous proliferation relative todermal cells in suspensions from normal subjects. The increasedproliferation is accompanied by the formation of clusters, which occursin psoriatic and not in normal cultures. The proliferation can onlypartly be accounted for by T cell proliferation. There appearsadditionally to be heterotypic or homotypic adhesion between the dermalcells, some of which may be macrophages and some of which may be stromalcells. To determine if LFA3TIP could block at least the T cell componentof the spontaneous proliferation, lesional dermal cells were plated insuspension. Approximately 2000 cpm was observed with dermal cells alone(normal controls generally exhibit between 200 and 1500 cpm). A limitingdose of IL-2 was then added at a concentration that should activate onlyT cells expressing the high affinity IL-2 receptor (10 units/ml on adaily basis×4 days). This boosted the counts to 4600 cpm (dermal cellsplus IL-2). Varying dilutions of LFA3TIP or human IgG were than added. Areduction in spontaneous dermal cell proliferation was seen with doseresponses between 0.03 and 0.003 μg/ml. Furthermore, the LFA3TIPcultures are consistently less proliferative than the human IgG controlcultures through the concentration of 0.01 μg/ml. These results providean interesting and important evidence of LFA3TIP activity on immunologicmechanisms occurring in fresh ex vivo psoriatic tissue.

[0153] 3. Proliferative Response of Allogeneic Mononuclear Cells toNormal Epidermal Cells

[0154] This experiment shows the ability of LFA3TIP to inhibitproliferation of allogeneic mononuclear cells to normal epidermal cells.Normal epidermal cells were prepared essentially as described forExample 1. MNC were prepared essentially as described for Example 1. Asshown in FIG. 5, epidermal-cells (EC) plus MNC demonstrated levels ofproliferation of approximately 22,000 cpm. Human IgG appears to beslightly enhancing between 12 and 0.3 μg/ml of LFA3TIP, relative to ECplus MNC alone. However, LFA3TP between 5 and 0.1 μg/ml, exerted a cleardose-responsive inhibition. Inhibition of Langerhans cell dependent Tcell activation models the type of activation that occurs in allergiccontact dermatitis, atopic dermatitis and mycosis fungoides type ofcutaneous T cell lymphoma.

[0155] 4. Proliferative Response of Autologous Mononuclear Cells toLesional Psoriatic Dermal Cells

[0156] FIG. 6demonstrates the response of autologous MNC to lesionaldermal cells prepared as a single cell suspension. Following dispasesplitting of the epidermis from the dermis, the dermis is digested bycollagenase, hyaluronidase, and DNase, and filtered through sequentialnylon mesh sizing filters. In this patient, the spontaneous level ofdermal cell proliferation was low, and induction of a dermal cell plusmononuclear cell reaction could be observed. This is a fairly complexsystem because the dermal cell preparation has a number of cell types,including macrophages, some neutrophils, antigen presenting cells,filbroblasts, mast cells, and endothelial cells, as well as Tlymphocytes. However, it is probably a reasonable approximation of thein vivo milieu as the infiltrating mononuclear cells first enter intoperivascular interstitium of the dermis in response to chemoattractivesignals present in psoriasis. Enhancement of the dermal cell-inducedproliferation occurred between 0.3 and 0.01 μg/ml of human IgG control.LFA3TIP inhibited this enhancement, and resulted in a reducedproliferative response between 1 and 0.03 μg/ml. In this dermal cellassay, the addition of IgG or removal of Fc IgG receptor bearing cells,specifically cells bearing the macrophage and neutrophil integrin CD11b,results in elevated spontaneous proliferation of dermal fibroblasts. Towhat degree the LFA3TIP inhibitory effect is upon T cell proliferationas opposed to complex FcγRIII-mediated macrophage antiproliferativeeffects on other cell types is not possible to determine. Regardless,the data clearly support a trend in both this and the previous dermalcell proliferation assays from psoriatic lesional skin which is in thetherapeutically beneficial direction for use of LFA3TIP.

[0157] 5. Proliferative Response of Allogeneic T lymphocytes to UVDamaged Epidermal Cells

[0158] Human subjects were exposed to 4 minimal erythemal doses ofultraviolet radiation, and 3 days later the epidermis was removed andprepared into an epidermal cell suspension. Suspensions were combinedwith resting, negatively selected CD4+ T lymphocytes which wereallogeneic to the sunburned donor. Two concentrations of UV-EC are shownin FIG. 7 A and B. Relative to T cells plus epidermal cells alone, or Tcells plus epidermal cells incubated with identical concentrations ofIgG, LFA3TIP incubation resulted in approximately 50% inhibition. Thecells were moderately free of antigen presenting cells (APC's), asevidenced by relatively minimal PHA proliferation, but were capable ofresponding as evidenced by responsiveness to PMA plus ionomycin. Thesedata demonstrate that inflammatory UV macrophages which migrate intosunburned skin use an LFA3TIP dependent mechanism to induce T cellactivation. Blockade of this process should be relevant to photoaging,in that repeated inflammatory activation in the skin is likelyresponsible for collagenase and elastase activation via lymphokineactivation. In addition, blockade of this signaling may reduce thegeneration of T suppressor cells which these UV induced macrophagesgenerate. These suppressor cells are the one responsible for tolerancein a contact sensitivity mode of UV-induced immunologic hostsusceptibility to UV carcinogenesis.

[0159] Deposits

[0160] Murine hybridoma cells and anti-LFA-3 antibodies useful in thepresent invention are exemplified by cultures deposited under theBudapest Treaty with American Type Culture Collection, Rockville, Md.,U.S.A., on Mar. 5, 1991, and identified as: Designation ATCC AccessionNo. 1E6 HB 10693 HC-1B11 HB 10694 7A6 HB 10695 8B8 HB 10696

[0161] A bacteriophage carrying a plasmid encoding transmembrane LFA-3was deposited under the Budapest Treaty with In Vitro International,Inc., Linthicum, Md., U.S.A., on May 28, 1987 under Accession No.IVI-10133. This deposit was transferred to American Type CultureCollection on Jun. 20, 1991 and identified as: Designation ATCCAccession No. λHT16[λgt10/LFA-3] 75107

[0162] E. coli transformed with a plasmid encoding PI-linked LFA-3 wasdeposited under the Budapest Treaty with In Vitro International, Inc. onJul. 22, 1988 under Accession No. IVI-10180. This deposit wastransferred to American Type Culture Collection on Jun. 20, 1991 andidentified as: Designation ATCC Accession No. p24 68788

[0163] Sequences

[0164] The following is a summary of the sequences set forth in theSequence Listing:

[0165] SEQ ID NO:1 DNA sequence of transmembrane LFA-3

[0166] SEQ ID NO:2 Amino acid sequence of transmembrane LFA-3

[0167] SEQ ID NO:3 DNA sequence of PI-linked LFA-3

[0168] SEQ ID NO:4 Amino acid sequence of PI-linked LFA-3

[0169] SEQ ID NO:5 DNA sequence of CD2

[0170] SEQ ID NO:6 Amino acid sequence of CD2

[0171] SEQ ID NO:7 DNA sequence of LFA3TIP

[0172] SEQ ID NO:8 Amino acid sequence of LFA3TIP

[0173] While we have hereinbefore described a number of embodiments ofthis invention, it is apparent that our basic embodiments can be alteredto provide other embodiments that utilize the processes of thisinvention. Therefore, it will be appreciated that the scope of thisinvention includes all alternative embodiments and variations which aredefined in the foregoing specification and by the claims appendedhereto; and the invention is not to be limited by the specificembodiments that have been presented herein by way of example.

1 8 753 base pairs nucleic acid single linear cDNA CDS 1..750sig_peptide 1..84 mat_peptide 85..750 misc_feature 1..750 /note= “Humantransmembrane LFA-3” misc_feature 646 /note= “Transmembrane domain” 1ATG GTT GCT GGG AGC GAC GCG GGG CGG GCC CTG GGG GTC CTC AGC GTG 48 MetVal Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val -28 -25 -20-15 GTC TGC CTG CTG CAC TGC TTT GGT TTC ATC AGC TGT TTT TCC CAA CAA 96Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln -10 -5 1ATA TAT GGT GTT GTG TAT GGG AAT GTA ACT TTC CAT GTA CCA AGC AAT 144 IleTyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn 5 10 15 20GTG CCT TTA AAA GAG GTC CTA TGG AAA AAA CAA AAG GAT AAA GTT GCA 192 ValPro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala 25 30 35 GAACTG GAA AAT TCT GAA TTC AGA GCT TTC TCA TCT TTT AAA AAT AGG 240 Glu LeuGlu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg 40 45 50 GTT TATTTA GAC ACT GTG TCA GGT AGC CTC ACT ATC TAC AAC TTA ACA 288 Val Tyr LeuAsp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr 55 60 65 TCA TCA GATGAA GAT GAG TAT GAA ATG GAA TCG CCA AAT ATT ACT GAT 336 Ser Ser Asp GluAsp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp 70 75 80 ACC ATG AAG TTCTTT CTT TAT GTG CTT GAG TCT CTT CCA TCT CCC ACA 384 Thr Met Lys Phe PheLeu Tyr Val Leu Glu Ser Leu Pro Ser Pro Thr 85 90 95 100 CTA ACT TGT GCATTG ACT AAT GGA AGC ATT GAA GTC CAA TGC ATG ATA 432 Leu Thr Cys Ala LeuThr Asn Gly Ser Ile Glu Val Gln Cys Met Ile 105 110 115 CCA GAG CAT TACAAC AGC CAT CGA GGA CTT ATA ATG TAC TCA TGG GAT 480 Pro Glu His Tyr AsnSer His Arg Gly Leu Ile Met Tyr Ser Trp Asp 120 125 130 TGT CCT ATG GAGCAA TGT AAA CGT AAC TCA ACC AGT ATA TAT TTT AAG 528 Cys Pro Met Glu GlnCys Lys Arg Asn Ser Thr Ser Ile Tyr Phe Lys 135 140 145 ATG GAA AAT GATCTT CCA CAA AAA ATA CAG TGT ACT CTT AGC AAT CCA 576 Met Glu Asn Asp LeuPro Gln Lys Ile Gln Cys Thr Leu Ser Asn Pro 150 155 160 TTA TTT AAT ACAACA TCA TCA ATC ATT TTG ACA ACC TGT ATC CCA AGC 624 Leu Phe Asn Thr ThrSer Ser Ile Ile Leu Thr Thr Cys Ile Pro Ser 165 170 175 180 AGC GGT CATTCA AGA CAC AGA TAT GCA CTT ATA CCC ATA CCA TTA GCA 672 Ser Gly His SerArg His Arg Tyr Ala Leu Ile Pro Ile Pro Leu Ala 185 190 195 GTA ATT ACAACA TGT ATT GTG CTG TAT ATG AAT GGT ATT CTG AAA TGT 720 Val Ile Thr ThrCys Ile Val Leu Tyr Met Asn Gly Ile Leu Lys Cys 200 205 210 GAC AGA AAACCA GAC AGA ACC AAC TCC AAT TGA 753 Asp Arg Lys Pro Asp Arg Thr Asn SerAsn 215 220 250 amino acids amino acid linear protein 2 Met Val Ala GlySer Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val -28 -25 -20 -15 Val CysLeu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln -10 -5 1 Ile TyrGly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn 5 10 15 20 ValPro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala 25 30 35 GluLeu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg 40 45 50 ValTyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr 55 60 65 SerSer Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp 70 75 80 ThrMet Lys Phe Phe Leu Tyr Val Leu Glu Ser Leu Pro Ser Pro Thr 85 90 95 100Leu Thr Cys Ala Leu Thr Asn Gly Ser Ile Glu Val Gln Cys Met Ile 105 110115 Pro Glu His Tyr Asn Ser His Arg Gly Leu Ile Met Tyr Ser Trp Asp 120125 130 Cys Pro Met Glu Gln Cys Lys Arg Asn Ser Thr Ser Ile Tyr Phe Lys135 140 145 Met Glu Asn Asp Leu Pro Gln Lys Ile Gln Cys Thr Leu Ser AsnPro 150 155 160 Leu Phe Asn Thr Thr Ser Ser Ile Ile Leu Thr Thr Cys IlePro Ser 165 170 175 180 Ser Gly His Ser Arg His Arg Tyr Ala Leu Ile ProIle Pro Leu Ala 185 190 195 Val Ile Thr Thr Cys Ile Val Leu Tyr Met AsnGly Ile Leu Lys Cys 200 205 210 Asp Arg Lys Pro Asp Arg Thr Asn Ser Asn215 220 723 base pairs nucleic acid single linear cDNA CDS 1..720sig_peptide 1..84 mat_peptide 85..720 misc_feature 1..720 /note= “HumanPI-linked LFA-3” misc_feature 568..720 /note= “Signal sequence forPI-linkage” 3 ATG GTT GCT GGG AGC GAC GCG GGG CGG GCC CTG GGG GTC CTCAGC GTG 48 Met Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu SerVal -28 -25 -20 -15 GTC TGC CTG CTG CAC TGC TTT GGT TTC ATC AGC TGT TTTTCC CAA CAA 96 Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe SerGln Gln -10 -5 1 ATA TAT GGT GTT GTG TAT GGG AAT GTA ACT TTC CAT GTA CCAAGC AAT 144 Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro SerAsn 5 10 15 20 GTG CCT TTA AAA GAG GTC CTA TGG AAA AAA CAA AAG GAT AAAGTT GCA 192 Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys ValAla 25 30 35 GAA CTG GAA AAT TCT GAA TTC AGA GCT TTC TCA TCT TTT AAA AATAGG 240 Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg40 45 50 GTT TAT TTA GAC ACT GTG TCA GGT AGC CTC ACT ATC TAC AAC TTA ACA288 Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr 5560 65 TCA TCA GAT GAA GAT GAG TAT GAA ATG GAA TCG CCA AAT ATT ACT GAT336 Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp 7075 80 ACC ATG AAG TTC TTT CTT TAT GTG CTT GAG TCT CTT CCA TCT CCC ACA384 Thr Met Lys Phe Phe Leu Tyr Val Leu Glu Ser Leu Pro Ser Pro Thr 8590 95 100 CTA ACT TGT GCA TTG ACT AAT GGA AGC ATT GAA GTC CAA TGC ATGATA 432 Leu Thr Cys Ala Leu Thr Asn Gly Ser Ile Glu Val Gln Cys Met Ile105 110 115 CCA GAG CAT TAC AAC AGC CAT CGA GGA CTT ATA ATG TAC TCA TGGGAT 480 Pro Glu His Tyr Asn Ser His Arg Gly Leu Ile Met Tyr Ser Trp Asp120 125 130 TGT CCT ATG GAG CAA TGT AAA CGT AAC TCA ACC AGT ATA TAT TTTAAG 528 Cys Pro Met Glu Gln Cys Lys Arg Asn Ser Thr Ser Ile Tyr Phe Lys135 140 145 ATG GAA AAT GAT CTT CCA CAA AAA ATA CAG TGT ACT CTT AGC AATCCA 576 Met Glu Asn Asp Leu Pro Gln Lys Ile Gln Cys Thr Leu Ser Asn Pro150 155 160 TTA TTT AAT ACA ACA TCA TCA ATC ATT TTG ACA ACC TGT ATC CCAAGC 624 Leu Phe Asn Thr Thr Ser Ser Ile Ile Leu Thr Thr Cys Ile Pro Ser165 170 175 180 AGC GGT CAT TCA AGA CAC AGA TAT GCA CTT ATA CCC ATA CCATTA GCA 672 Ser Gly His Ser Arg His Arg Tyr Ala Leu Ile Pro Ile Pro LeuAla 185 190 195 GTA ATT ACA ACA TGT ATT GTG CTG TAT ATG AAT GGT ATG TATGCT TTT 720 Val Ile Thr Thr Cys Ile Val Leu Tyr Met Asn Gly Met Tyr AlaPhe 200 205 210 TAA 723 240 amino acids amino acid linear protein 4 MetVal Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val -28 -25 -20-15 Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln -10-5 1 Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn 510 15 20 Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala25 30 35 Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg40 45 50 Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr55 60 65 Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp70 75 80 Thr Met Lys Phe Phe Leu Tyr Val Leu Glu Ser Leu Pro Ser Pro Thr85 90 95 100 Leu Thr Cys Ala Leu Thr Asn Gly Ser Ile Glu Val Gln Cys MetIle 105 110 115 Pro Glu His Tyr Asn Ser His Arg Gly Leu Ile Met Tyr SerTrp Asp 120 125 130 Cys Pro Met Glu Gln Cys Lys Arg Asn Ser Thr Ser IleTyr Phe Lys 135 140 145 Met Glu Asn Asp Leu Pro Gln Lys Ile Gln Cys ThrLeu Ser Asn Pro 150 155 160 Leu Phe Asn Thr Thr Ser Ser Ile Ile Leu ThrThr Cys Ile Pro Ser 165 170 175 180 Ser Gly His Ser Arg His Arg Tyr AlaLeu Ile Pro Ile Pro Leu Ala 185 190 195 Val Ile Thr Thr Cys Ile Val LeuTyr Met Asn Gly Met Tyr Ala Phe 200 205 210 1056 base pairs nucleic acidsingle linear cDNA CDS 1..1053 sig_peptide 1..72 mat_peptide 73..1053misc_feature 1..1053 /note= “Human CD2” misc_feature 628..702 /note=“Transmembrane domain” 5 ATG AGC TTT CCA TGT AAA TTT GTA GCC AGC TTC CTTCTG ATT TTC AAT 48 Met Ser Phe Pro Cys Lys Phe Val Ala Ser Phe Leu LeuIle Phe Asn -24 -20 -15 -10 GTT TCT TCC AAA GGT GCA GTC TCC AAA GAG ATTACG AAT GCC TTG GAA 96 Val Ser Ser Lys Gly Ala Val Ser Lys Glu Ile ThrAsn Ala Leu Glu -5 1 5 ACC TGG GGT GCC TTG GGT CAG GAC ATC AAC TTG GACATT CCT AGT TTT 144 Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp IlePro Ser Phe 10 15 20 CAA ATG AGT GAT GAT ATT GAC GAT ATA AAA TGG GAA AAAACT TCA GAC 192 Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys ThrSer Asp 25 30 35 40 AAG AAA AAG ATT GCA CAA TTC AGA AAA GAG AAA GAG ACTTTC AAG GAA 240 Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu Thr PheLys Glu 45 50 55 AAA GAT ACA TAT AAG CTA TTT AAA AAT GGA ACT CTG AAA ATTAAG CAT 288 Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile LysHis 60 65 70 CTG AAG ACC GAT GAT CAG GAT ATC TAC AAG GTA TCA ATA TAT GATACA 336 Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp Thr75 80 85 AAA GGA AAA AAT GTG TTG GAA AAA ATA TTT GAT TTG AAG ATT CAA GAG384 Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys Ile Gln Glu 9095 100 AGG GTC TCA AAA CCA AAG ATC TCC TGG ACT TGT ATC AAC ACA ACC CTG432 Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile Asn Thr Thr Leu 105110 115 120 ACC TGT GAG GTA ATG AAT GGA ACT GAC CCC GAA TTA AAC CTG TATCAA 480 Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu Asn Leu Tyr Gln125 130 135 GAT GGG AAA CAT CTA AAA CTT TCT CAG AGG GTC ATC ACA CAC AAGTGG 528 Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val Ile Thr His Lys Trp140 145 150 ACC ACC AGC CTG AGT GCA AAA TTC AAG TGC ACA GCA GGG AAC AAAGTC 576 Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys Thr Ala Gly Asn Lys Val155 160 165 AGC AAG GAA TCC AGT GTC GAG CCT GTC AGC TGT CCA GAG AAA GGTCTG 624 Ser Lys Glu Ser Ser Val Glu Pro Val Ser Cys Pro Glu Lys Gly Leu170 175 180 GAC ATC TAT CTC ATC ATT GGC ATA TGT GGA GGA GGC AGC CTC TTGATG 672 Asp Ile Tyr Leu Ile Ile Gly Ile Cys Gly Gly Gly Ser Leu Leu Met185 190 195 200 GTC TTT GTG GCA CTG CTC GTT TTC TAT ATC ACC AAA AGG AAAAAA CAG 720 Val Phe Val Ala Leu Leu Val Phe Tyr Ile Thr Lys Arg Lys LysGln 205 210 215 AGG AGT CGG AGA AAT GAT GAG GAG CTG GAG ACA AGA GCC CACAGA GTA 768 Arg Ser Arg Arg Asn Asp Glu Glu Leu Glu Thr Arg Ala His ArgVal 220 225 230 GCT ACT GAA GAA AGG GGC CGG AAG CCC CAC CAA ATT CCA GCTTCA ACC 816 Ala Thr Glu Glu Arg Gly Arg Lys Pro His Gln Ile Pro Ala SerThr 235 240 245 CCT CAG AAT CCA GCA ACT TCC CAA CAT CCT CCT CCA CCA CCTGGT CAT 864 Pro Gln Asn Pro Ala Thr Ser Gln His Pro Pro Pro Pro Pro GlyHis 250 255 260 CGT TCC CAG GCA CCT AGT CAT CGT CCC CCG CCT CCT GGA CACCGT GTT 912 Arg Ser Gln Ala Pro Ser His Arg Pro Pro Pro Pro Gly His ArgVal 265 270 275 280 CAG CAC CAG CCT CAG AAG AGG CCT CCT GCT CCG TCG GGCACA CAA GTT 960 Gln His Gln Pro Gln Lys Arg Pro Pro Ala Pro Ser Gly ThrGln Val 285 290 295 CAC CAG CAG AAA GGC CCG CCC CTC CCC AGA CCT CGA GTTCAG CCA AAA 1008 His Gln Gln Lys Gly Pro Pro Leu Pro Arg Pro Arg Val GlnPro Lys 300 305 310 CCT CCC CAT GGG GCA GCA GAA AAC TCA TTG TCC CCT TCCTCT AAT 1053 Pro Pro His Gly Ala Ala Glu Asn Ser Leu Ser Pro Ser Ser Asn315 320 325 TAA 1056 351 amino acids amino acid linear protein 6 Met SerPhe Pro Cys Lys Phe Val Ala Ser Phe Leu Leu Ile Phe Asn -24 -20 -15 -10Val Ser Ser Lys Gly Ala Val Ser Lys Glu Ile Thr Asn Ala Leu Glu -5 1 5Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro Ser Phe 10 15 20Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys Thr Ser Asp 25 30 3540 Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu Thr Phe Lys Glu 45 5055 Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile Lys His 60 6570 Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp Thr 75 8085 Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys Ile Gln Glu 90 95100 Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile Asn Thr Thr Leu 105110 115 120 Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu Asn Leu TyrGln 125 130 135 Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val Ile Thr HisLys Trp 140 145 150 Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys Thr Ala GlyAsn Lys Val 155 160 165 Ser Lys Glu Ser Ser Val Glu Pro Val Ser Cys ProGlu Lys Gly Leu 170 175 180 Asp Ile Tyr Leu Ile Ile Gly Ile Cys Gly GlyGly Ser Leu Leu Met 185 190 195 200 Val Phe Val Ala Leu Leu Val Phe TyrIle Thr Lys Arg Lys Lys Gln 205 210 215 Arg Ser Arg Arg Asn Asp Glu GluLeu Glu Thr Arg Ala His Arg Val 220 225 230 Ala Thr Glu Glu Arg Gly ArgLys Pro His Gln Ile Pro Ala Ser Thr 235 240 245 Pro Gln Asn Pro Ala ThrSer Gln His Pro Pro Pro Pro Pro Gly His 250 255 260 Arg Ser Gln Ala ProSer His Arg Pro Pro Pro Pro Gly His Arg Val 265 270 275 280 Gln His GlnPro Gln Lys Arg Pro Pro Ala Pro Ser Gly Thr Gln Val 285 290 295 His GlnGln Lys Gly Pro Pro Leu Pro Arg Pro Arg Val Gln Pro Lys 300 305 310 ProPro His Gly Ala Ala Glu Asn Ser Leu Ser Pro Ser Ser Asn 315 320 325 1050base pairs nucleic acid single linear cDNA CDS 1..1041 sig-peptide 1..84mat_peptide 85..1041 misc-feature 85..1041 /note- “LFA3TIP” misc-feature360..361 /note- “LFA-3/IgG fusion point” 7 ATG GTT GCT GGG AGC GAC GCGGGG CGG GCC CTG GGG GTC CTC AGC GTG 48 Met Val Ala Gly Ser Asp Ala GlyArg Ala Leu Gly Val Leu Ser Val -28 -25 -20 -15 GTC TGC CTG CTG CAC TGCTTT GGT TTC ATC AGC TGT TTT TCC CAA CAA 96 Val Cys Leu Leu His Cys PheGly Phe Ile Ser Cys Phe Ser Gln Gln -10 -5 1 ATA TAT GGT GTT GTG TAT GGGAAT GTA ACT TTC CAT GTA CCA AGC AAT 144 Ile Tyr Gly Val Val Tyr Gly AsnVal Thr Phe His Val Pro Ser Asn 5 10 15 20 GTG CCT TTA AAA GAG GTC CTATGG AAA AAA CAA AAG GAT AAA GTT GCA 192 Val Pro Leu Lys Glu Val Leu TrpLys Lys Gln Lys Asp Lys Val Ala 25 30 35 GAA CTG GAA AAT TCT GAA TTC AGAGCT TTC TCA TCT TTT AAA AAT AGG 240 Glu Leu Glu Asn Ser Glu Phe Arg AlaPhe Ser Ser Phe Lys Asn Arg 40 45 50 GTT TAT TTA GAC ACT GTG TCA GGT AGCCTC ACT ATC TAC AAC TTA ACA 288 Val Tyr Leu Asp Thr Val Ser Gly Ser LeuThr Ile Tyr Asn Leu Thr 55 60 65 TCA TCA GAT GAA GAT GAG TAT GAA ATG GAATCG CCA AAT ATT ACT GAT 336 Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu SerPro Asn Ile Thr Asp 70 75 80 ACC ATG AAG TTC TTT CTT TAT GTC GAC AAA ACTCAC ACA TGC CCA CCG 384 Thr Met Lys Phe Phe Leu Tyr Val Asp Lys Thr HisThr Cys Pro Pro 85 90 95 100 TGC CCA GCA CCT GAA CTC CTG GGG GGA CCG TCAGTC TTC CTC TTC CCC 432 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser ValPhe Leu Phe Pro 105 110 115 CCA AAA CCC AAG GAC ACC CTC ATG ATC TCC CGGACC CCT GAG GTC ACA 480 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg ThrPro Glu Val Thr 120 125 130 TGC GTG GTG GTG GAC GTG AGC CAC GAA GAC CCTGAG GTC AAG TTC AAC 528 Cys Val Val Val Asp Val Ser His Glu Asp Pro GluVal Lys Phe Asn 135 140 145 TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCCAAG ACA AAG CCG CGG 576 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala LysThr Lys Pro Arg 150 155 160 GAG GAG CAG TAC AAC AGC ACG TAC CGG GTG GTCAGC GTC CTC ACC GTC 624 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val SerVal Leu Thr Val 165 170 175 180 CTG CAC CAG GAC TGG CTG AAT GGC AAG GAGTAC AAG TGC AAG GTC TCC 672 Leu His Gln Asp Trp Leu Asn Gly Lys Glu TyrLys Cys Lys Val Ser 185 190 195 AAC AAA GCC CTC CCA GCC CCC ATC GAG AAAACC ATC TCC AAA GCC AAA 720 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys ThrIle Ser Lys Ala Lys 200 205 210 GGG CAG CCC CGA GAA CCA CAG GTG TAC ACCCTG CCC CCA TCC CGG GAT 768 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr LeuPro Pro Ser Arg Asp 215 220 225 GAG CTG ACC AAG AAC CAG GTC AGC CTG ACCTGC CTG GTC AAA GGC TTC 816 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr CysLeu Val Lys Gly Phe 230 235 240 TAT CCC AGC GAC ATC GCC GTG GAG TGG GAGAGC AAT GGG CAG CCG GAG 864 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu SerAsn Gly Gln Pro Glu 245 250 255 260 AAC AAC TAC AAG ACC ACG CCT CCC GTGCTG GAC TCC GAC GGC TCC TTC 912 Asn Asn Tyr Lys Thr Thr Pro Pro Val LeuAsp Ser Asp Gly Ser Phe 265 270 275 TTC CTC TAC AGC AAG CTC ACC GTG GACAAG AGC AGG TGG CAG CAG GGG 960 Phe Leu Tyr Ser Lys Leu Thr Val Asp LysSer Arg Trp Gln Gln Gly 280 285 290 AAC GTC TTC TCA TGC TCC GTG ATG CATGAG GCT CTG CAC AAC CAC TAC 1008 Asn Val Phe Ser Cys Ser Val Met His GluAla Leu His Asn His Tyr 295 300 305 ACG CAG AAG AGC CTC TCC CTG TCT CCGGGT AAA TGAGTGCGG 1050 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 310315 347 amino acids amino acid linear protein 8 Met Val Ala Gly Ser AspAla Gly Arg Ala Leu Gly Val Leu Ser Val -28 -25 -20 -15 Val Cys Leu LeuHis Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln -10 -5 1 Ile Tyr Gly ValVal Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn 5 10 15 20 Val Pro LeuLys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala 25 30 35 Glu Leu GluAsn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg 40 45 50 Val Tyr LeuAsp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr 55 60 65 Ser Ser AspGlu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp 70 75 80 Thr Met LysPhe Phe Leu Tyr Val Asp Lys Thr His Thr Cys Pro Pro 85 90 95 100 Cys ProAla Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 105 110 115 ProLys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 120 125 130Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 135 140145 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 150155 160 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val165 170 175 180 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys LysVal Ser 185 190 195 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile SerLys Ala Lys 200 205 210 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu ProPro Ser Arg Asp 215 220 225 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr CysLeu Val Lys Gly Phe 230 235 240 Tyr Pro Ser Asp Ile Ala Val Glu Trp GluSer Asn Gly Gln Pro Glu 245 250 255 260 Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe 265 270 275 Phe Leu Tyr Ser Lys Leu ThrVal Asp Lys Ser Arg Trp Gln Gln Gly 280 285 290 Asn Val Phe Ser Cys SerVal Met His Glu Ala Leu His Asn His Tyr 295 300 305 Thr Gln Lys Ser LeuSer Leu Ser Pro Gly Lys 310 315

We claim:
 1. A method of preventing or treating skin conditionscharacterized by increased T cell activation and abnormal antigenpresentation in the dermis and epidermis comprising the step ofadministering to a mammal, including a human, an inhibitor of theCD2/LFA-3 interaction.
 2. The method according to claim 1, wherein thecondition is selected from the group consisting of atopic dermatitis,cutaneous T cell lymphoma such as mycosis fungoides, allergic andirritant contact dermatitis, lichen planus, alopecia areata, pyodermagangrenosum, vitiligo, ocular cicatricial pemphigoid, and urticaria. 3.The method according to claim 1, wherein the condition is psoriasis. 4.The method according to claim 1, wherein the inhibitor is selected fromthe group consisting of anti-LFA-3 antibody homologs, and soluble CD2polypeptides.
 5. The method according to claim 1, wherein the inhibitoris selected from the group consisting of anti-CD2 antibody homologs andsoluble LFA-3 polypeptides.
 6. The method according to claim 5, whereinsaid soluble LFA-3 polypeptide is a soluble LFA-3 polypeptide fused toall or part of an immunoglobulin heavy chain region and all or part of aheavy chain constant region.
 7. The method according to claim 6, whereinsaid soluble LFA-3 polypeptide is LFA3TIP.
 8. The method according toclaim 4, wherein the inhibitor is an anti-LFA-3 antibody homolog.
 9. Themethod according to claim 5, wherein the inhibitor is an anti-CD2antibody homolog.
 10. The method according to claim 8, wherein theinhibitor is a monoclonal anti-LFA-3 antibody.
 11. The method accordingto claim 9, wherein the inhibitor is a monoclonal anti-CD2 antibody. 12.The method according to claim 10, wherein the inhibitor is a monoclonalanti-LFA-3 antibody produced by a hybridoma selected from the group ofhybridomas having Accession Nos. ATCC HB 10693 (1E6), ATCC HB 10694(HC-1B11), ATCC HB 10695 (7A6), and ATCC HB 10696 (8B8) or is monoclonalantibody TS2/9.
 13. The method according to claim 12, wherein themonoclonal anti-LFA-3 antibody is produced by a hybridoma selected fromthe group of hybridomas having Accession Nos. ATCC HB 10695 (7A6) andATCC HB 10693 (1E6).
 14. The method according to claim 8, wherein theinhibitor is a chimeric recombinant anti-LFA-3 antibody homolog.
 15. Themethod according to claim 9, wherein the inhibitor is a chimericrecombinant anti-CD2 antibody homolog.
 16. The method according to claim8, wherein the inhibitor is a humanized recombinant anti-LFA-3 antibodyhomolog.
 17. The method according to claim 9, wherein the inhibitor is ahumanized recombinant anti-CD2 antibody homolog.
 18. The methodaccording to claim 8, wherein the inhibitor is selected from the groupconsisting of Fab fragments, Fab′ fragments, F(ab′) 2 fragments, F(v)fragments and intact immunoglobulin heavy chains of an anti-LFA-3antibody homolog.
 19. The method according to claim 9, wherein theinhibitor is selected from the group consisting of Fab fragments, Fab′fragments, F(ab′) 2 fragments, F(v) fragments and intact immunoglobulinheavy chains of an anti-CD2 antibody homolog.
 20. The method accordingto claim 5, wherein the inhibitor is a soluble LFA-3 polypeptide. 21.The method according to claim 4, wherein the inhibitor is a soluble CD2polypeptide.
 22. The method according to claim 20, wherein the inhibitoris a soluble LFA-3 polypeptide selected from the group of polypeptidesconsisting of AA₁-AA₉₂ of SEQ ID NO:2, AA₁-AA₈₀ of SEQ ID NO:2,AA₅₀-AA₆₅ of SEQ ID NO:2, and AA₂₀-AA₈₀ of SEQ ID NO:2.
 23. The methodaccording to claim 1, wherein the mammal is a human.
 24. The methodaccording to claim 1, wherein the inhibitor is administered at a dosebetween about 0.001 and about 50 mg inhibitor per kg body weight. 25.The method according to claim 24, wherein the inhibitor is administeredat a dose between about 0.01 and about 10 mg inhibitor per kg bodyweight.
 26. The method according to claim 24, wherein the inhibitor isadministered at a dose between about 0.1 and about 4 mg inhibitor per kgbody weight.
 27. The method according to claim 24, wherein the dose isadministered once to three times per week.
 28. The method according toclaim 24, wherein the dose is administered once to three times per day.29. The method according to claim 28, wherein the dose is administeredabout one to three times daily for between 3 and 7 days.
 30. The methodaccording to claim 29, wherein the dose is administered about one tothree times daily for between 3 and 7 days on a monthly basis.
 31. Themethod according to claim 1, wherein the inhibitor is administeredintravenously, intramuscularly, subcutaneously, intra-articularly,intrathecally, periostally, intratumorally, intralesionally,perilesionally by infusion, orally, topically or by inhalation.
 32. Themethod according to claim 31, wherein the inhibitor is administeredintramuscularly, intravenously or subcutaneously.
 33. The methodaccording to claim 4, wherein the inhibitor is linked to one or moremembers independently selected from the group consisting of anti-LFA-3antibody homologs, soluble CD2 polypeptides, cytotoxic agents andpharmaceutical agents.
 34. The method according to claim 5, wherein theinhibitor is linked to one or more members independently selected fromthe group consisting of anti-CD2 antibody homologs, soluble LFA-3polypeptides, cytotoxic agents and pharmaceutical agents.
 35. The methodaccording to claim 34, wherein the inhibitor is a polypeptide consistingof a soluble LFA-3 polypeptide linked to an immunoglobulin hinge andheavy chain constant region or portions thereof.
 36. The methodaccording to claim 35, wherein said polypeptide is LFA3TIP.
 37. Themethod according to claim 1, wherein the condition is UV damage.
 38. Amethod of preventing or treating skin conditions characterized byincreased T cell activation and abnormal antigen presentation in thedermis and epidermis comprising the step of administering to a mammal,including a human, a composition comprising an agent which binds toLFA-3 or CD2 chosen from the group of CD2 polypeptides, LFA-3polypeptides, anti-CD2 antibody homologs, and anti-LFA-3 antibodyhomologs.
 39. The method of claim 38, wherein said agent is a CD2polypeptide.
 40. The method of claim 39, wherein said CD2 polypeptide isa soluble CD2 polypeptide.
 41. The method of claim 38, wherein saidagent is an LFA-3 polypeptide.
 42. The method of claim 41, wherein saidLFA-3 polypeptide is a soluble LFA-3 polypeptide.
 43. The method ofclaim 42, wherein said soluble LFA-3 polypeptide is a soluble LFA-3polypeptide fused to all or part of an immunoglobulin heavy chain regionand all or part of a heavy chain constant region.
 44. The method ofclaim 43, wherein said soluble LFA-3 polypeptide is LFA3TIP.
 45. Themethod of claim 38, wherein said agent is an anti-CD2 antibody homolog.46. The method of claim 45, wherein said anti-CD2 antibody homolog is ahumanized recombinant anti-CD2 antibody homolog or chimeric recombinantanti-CD2 antibody homolog.
 47. The method of claim 38, wherein saidagent is an anti-LFA-3 antibody homolog.
 48. The method of claim 47,wherein said anti-LFA-3 antibody homolog is a humanized recombinantanti-LFA-3 antibody homolog or chimeric recombinant anti-LFA-3 antibodyhomolog.